Diagnostic tumor markers, drug screening for tumorigenesis inhibition, and compositions and methods for treatment of cancer

ABSTRACT

The invention provides a series of compositions, methods, kits, articles and species associated primarily with the diagnosis and/or treatment of cell proliferation, specifically cancer. Cell proliferation associated with aberrant expression of MUC1 is particularly focused upon. Mechanisms associated with MUC1 cell proliferation are discussed.

RELATED APPLICATIONS

[0001] This non-provisional application claims the benefit under Title35, U.S.C. §119(e) of co-pending U.S. provisional application No.60/253,361, filed Nov. 27, 2000, U.S. provisional application No.60/255,370, filed Dec. 13, 2000, U.S. provisional application No.60/256,027, filed Dec. 15, 2000, U.S. provisional application No.60/258,157, filed Dec. 22, 2000, U.S. provisional application No.60/259,615, filed Jan. 3, 2001, U.S. provisional application No.60/260,186, filed Jan. 5, 2001, U.S. provisional application No.60/266,169, filed Feb. 2, 2001, U.S. provisional application No.60/289,444, filed May 7, 2001, U.S. provisional application No.60/266,929, filed Feb. 6, 2001, U.S. provisional application No.60/278,093, filed Mar. 23, 2001, U.S. provisional application No.60/294,887, filed May 31, 2001, and U.S. provisional application No.60/298,272, filed Jun. 14, 2001, each of which is incorporated byreference herein.

FIELD OF THE INVENTION

[0002] The invention relates to assays using shed cell surface receptorinterchain binding regions and cleavage products for cancer diagnosis,and for the evaluation of cancer treatment and using the portion of thereceptor that remains on the cell as a molecular target for cancertherapeutics.

BACKGROUND OF THE INVENTION

[0003] Many of the biomolecular interactions that promote tumorigenesisinvolve cell surface proteins that mediate both intra- and intercellularsignaling. “Tumor markers” are proteins on the surface of a cell thatare exclusively expressed, over-expressed or show an altered expressionpattern as a result of transformation to a neoplastic state. The surfaceconcentration of certain tumor markers has been correlated to theprogression of cancer. For example, the interaction between the cellsurface receptor αVβ3 and the cell adhesion molecule vitronectin hasbeen implicated in angiogenesis (Varner J, Cheresh D: Integrins andcancer. Curr Opin Cell Biol, 1996, 8(5): 724-730; Vailhe B, Ronot X,Tracqui P, Usson Y, Tracqui L: In vitro angiogenesis is modulated by themechanical properties of fibrin gels and is related to αVβ3 integrinlocalization. In Vitro Cell Dev Biol Anim, 1997, 33(10): 763-773; HortonM: The aVb3 integrin “vitronectin receptor”. Int J Biochem Cell Biol,1997, 29(5): 721-725) and the increased concentration of αVβ3 onmelanoma cells has been correlated with poor prognosis (Hieken T,Farolan M, Ronan S, Shilkaitis A, Wild L, Das Gupta T: β3 integrinexpression in melanoma predicts subsequent metastasis. J Surg Res, 1996,63(1): 169-173).

[0004] Cell surface receptors, that have been linked to cancer, make upan important class of therapeutic targets. Many pharmaceutical companiesare actively involved in screening drug libraries for compounds thatbind to and block these cell surface receptors. For example, animportant drug used to treat breast cancer is Herceptin (Pegram M,Lipton A, Hayes D, Webber B, Baselga J, Tripathy D, Baly D, Baughman S,Twaddell T, Glaspy J, Slamon D: Phase II study of receptor-enhancedchemosensitivity using recombinant humanized anti-p185 Her2/neumonoclonal antibody plus cisplatin, in patients withHer2/neu-overexpressing metastatic breast cancer refractory tochemotherapy treatment, J Clin Oncol, 1998, 16(8): 2659-2671). This drugbinds to and blocks HER2/neu (Ross J, Fletcher J: review, The Her2/neuoncogene in breast cancer: prognostic factor, predictive factor, andtarget for therapy. Stem Cells, 1998, 16(6): 413-428) which is a cellsurface receptor that is over-expressed on 30% of breast tumors.

[0005] Another cell surface receptor, called MUC1 (Treon S, Mollick J,Urashima M, Teoh G, Chauhan D, Ogata A, Raje N, Hilgers J, Nadler L,Belch A, Pilarski L and Anderson K: MUC1 core protein is expressed onmultiple myeloma cells and is induced by dexamethasone. Blood, 1999,93(4): 1287-1298), is especially interesting since it is aberrantlyexpressed on many human tumors, including 80% of breast tumors, and on asignificant percentage of prostate, lung, ovarian, colorectal andperhaps brain, cancers. On healthy secretory epithelium, MUC1 isclustered at the apical border and is not expressed over other portionsof the cell. However, in tumor cells, the receptor is homogeneouslyover-expressed over the entire cell surface (Kufe D., Inghirami G., AbeM., Hayes D, Justi-Wheeler H, Schlom J: Differential reactivity of anovel monoclonal antibody (DF3) with human malignant versus benignbreast tumors. Hybridoma, 1984, 3: 223-232), rather than just at theapical border. It is also known that women with breast cancer haveelevated levels of shed MUC1 receptor in their blood stream.Extracellular portions of the MUC1 receptor are cleaved or “shed”, by atleast one enzyme, and released into the blood stream. Levels of shedMUC1 receptor in serum are measured to track breast cancer patients forrecurrence. However, the method is too variable and insensitive to beused as a general diagnostic.

[0006] Until now, the mechanistic link between the MUC1 receptor andtumorigenesis has not been understood. Attempts to correlate the numberof repeat units, which varies from person to person, and susceptibilityto cancer failed. Investigations of a possible connection, betweenglycosylation of the MUC1 receptor and cancer, produced conflictingresults. Importantly, until now, a functional ligand(s) for theextracellular portion of the MUC1 receptor has not been identified.

[0007] Absent an understanding of the mechanism of the MUC1 receptor,and how it triggers tumorigenesis, it has not been possible to design oridentify therapeutics that interfere with the disease-associatedfunction of this receptor. Indeed, currently there is no drug in use or,to our knowledge, in clinical trials that is known to target the MUC1receptor.

[0008] The present invention describes discoveries that elucidatecritical aspects of the mechanism by which MUC1 triggers cellproliferation and tumorigenesis. These discoveries provide novelmolecular targets for drug screening assays which the inventors haveused to identify compounds that inhibit the MUC1-dependenttumorigenesis. These discoveries also enable an early diagnostic assay.

SUMMARY OF THE INVENTION

[0009] The present invention provides a variety of kits, methods,compositions, peptide species and articles associated with cellproliferation, specifically cancer. The invention involves primarilytechniques and components for the diagnosis and treatment of cancer.

[0010] In one aspect, the invention provides a series of kits. One kitincludes a first article having a surface, and a peptide sequenceimmobilized relative to or adapted to be immobilized relative to thesurface. The peptide sequence includes a portion of a cell surfacereceptor that interacts with an activating ligand such as a growthfactor to promote cell proliferation. Also included in the kit is acandidate drug for affecting the ability of the peptide sequence to bindto other identical peptide sequences in the presence of the activatingligand. The portion includes enough of the cell surface receptor tointeract with the activating ligand and the portion is free ofinterchain binding region to the extent necessary to prevent spontaneousbinding between the portions.

[0011] Another kit of the invention comprises a species able to becomeimmobilized relative to a shed cell surface receptor interchain bindingregion, and a signaling entity immobilized relative to or adapted to beimmobilized relative to the species.

[0012] Another kit of the invention comprises a species able to bind toa portion of a cell surface receptor that remains attached to the cellsurface after shedding of a cell surface receptor interchain bindingregion, and a signaling entity immobilized relative to or adapted to beimmobilized relative to the species.

[0013] Another kit of the invention comprises a species able to bind toa portion of a cell surface receptor that includes the interchainbinding region, and a signaling entity immobilized relative to oradapted to be immobilized relative to the species.

[0014] Another kit of the invention comprises an article (which can be aparticle), and at least a fragment of the sequence that corresponds tothat portion of a cell surface receptor that interacts with anactivating ligand such as a growth factor to promote cell proliferation,the fragment being detached from any cell, fastened to or adapted to befastened to the article.

[0015] Another kit of the invention comprises an article having asurface, and a biomolecule that binds to a portion of a cell surfacereceptor that interacts with an activating ligand such as a growthfactor to promote cell proliferation. The biomolecule is fastened to oradapted to be fastened to the surface of the article.

[0016] In another aspect, the invention provides a series of methods.One method comprises providing a peptide including a portion of a cellsurface receptor that interacts with an activating ligand such as agrowth factor to promote cell proliferation, exposing the peptide to acandidate drug for affecting the ability of the activating ligand tointeract with the peptide, and to the activating ligand, and determiningthe ability of the candidate drug to prevent interaction of theactivating ligand with the peptide. The portion includes enough of thecell surface receptor to interact with the activating ligand and theportion is free of interchain binding region to the extent necessary toprevent spontaneous binding between portions.

[0017] Another method of the invention involves treating a subjecthaving cancer or being at risk for developing cancer, the methodcomprises administering to the subject an agent that reduces cleavage ofa cell surface receptor.

[0018] Another method of the invention for treating a subject havingcancer or at risk for developing cancer comprises administering to thesubject an agent that reduces cleavage of a cell surface receptorinterchain binding region from the cell surface.

[0019] Another method of the invention comprises determining an amountof cleavage of a cell surface receptor interchain binding region from acell surface, and evaluating indication of cancer or potential forcancer based upon the determining step.

[0020] Another method of the invention comprises determining a site ofcleavage of a cell surface receptor in a sample from a subject, andevaluating an indication of cancer or potential for cancer based uponthe determining step.

[0021] Another method of the invention involves determining a cleavagesite of a cell surface. The method comprises contacting a cell with anagent that binds specifically to one potential cell surface receptorcleavage site and another agent that binds specifically to anotherpotential cell surface receptor cleavage site. The ratio of binding ofthe two agents to the cell surface is compared in the method.

[0022] Another method of the invention comprises determining a firstamount of cleavage of a cell surface receptor interchain binding regionfrom a cell surface of a sample from a subject. A second amount ofcleavage of cell surface receptor interchain binding region from a cellsurface of a sample from the subject is also determined, and the firstamount is compared to the second amount.

[0023] Another method of the invention involves treating a subject toreduce the risk of or progression of cancer. The method comprisesadministering to a subject, who is known to be at risk for cancer or isdiagnosed with cancer, an agent for inhibiting interaction of anactivating ligand with a portion of a cell surface receptor thatinteracts with the activating ligand to promote cell proliferation.

[0024] Another method of the invention involves treating a subject toreduce the risk of or progression of cancer. The method comprisesadministering to a subject, who is known to be at risk of cancer or isdiagnosed with cancer, an agent for preventing clustering of portions ofcell surface receptors that interact with an activating ligand such as agrowth factor to promote cell proliferation.

[0025] Another method of the invention comprises exposing a ligandcapable of binding with a portion of a cell surface receptor thatremains attached to the cell after shedding of the cell surface receptorinterchain binding region, and an agent capable of blocking thisbinding, to a candidate drug for disruption of interaction between theligand and the agent. Disruption of the interaction by the candidatedrug is determined.

[0026] Another method of the invention comprises exposing a portion of acell surface receptor that remains attached to the cell surface aftershedding of a cell surface receptor interchain binding region which iscapable of binding with a ligand, and an agent capable of blocking thisbinding, to a candidate drug for disruption of interaction between theportion and the agent, and determining disruption of the interaction bythe candidate drug.

[0027] Another method of the invention comprises exposing a syntheticdrug, and a biological target of the synthetic drug, to a candidate drugwhich may interact with a biological target to a degree greater than theinteraction between the synthetic drug and the target, and determiningdisruption of the interaction by the candidate drug.

[0028] Another method involves diagnosing a physiological stateindicative of cancer or potential for cancer. The method comprisesdetermining a specific cleavage site of MUC1 distinguishable from adifferent cleavage state of MUC1.

[0029] Another method of the invention involves treating a subjecthaving a cancer characterized by the aberrant expression of MUC1,comprising administering to the subject etomoxir in an amount effectiveto reduce tumor growth.

[0030] Another method of the invention involves treating a subjecthaving a cancer characterized by the aberrant expression of MUC1,comprising administering to the subject L-α-methyl-dopa in an amounteffective to reduce tumor growth.

[0031] Another method of the invention for treating a subject havingcancer characterized by the aberrant expression of MUC1, comprisesadministering to the subject calcimycin in an amount effective to reducetumor growth.

[0032] Another method for treating a subject having a cancercharacterized by the aberrant expression of MUC1, comprisesadministering to the subject butylindazole in an amount effective toreduce tumor growth.

[0033] In another aspect, the invention provides compositions. Onecomposition of the invention comprises at least a portion of a shed cellsurface receptor interchain binding region, and a signaling entityimmobilized relative to or adapted to be immobilized relative to theportion.

[0034] The invention also provides peptide species. One peptide speciesof the invention comprises at least a fragment of a sequence thatcorresponds to that portion of a cell surface receptor that interactswith an activating ligand such as a growth factor to promote cellproliferation, the portion being detached from any cell, and an affinitytag.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is a schematic illustration of the MUC1 receptor;

[0036]FIG. 2 (color) is a colloid-based, color change binding experimentthat shows which portions of soluble MUC1 bind to each other orself-aggregate;

[0037]FIG. 3 (color) is a colloid-based color change experiment in whichthe ability of peptides to self-aggregate was used to help determine aboundary between a portion of the MUC1 receptor that self-aggregates thecell-proximal portion that does not; results imply a disease-relatedcleavage site on the MUC1 receptor;

[0038]FIG. 4 is a graph of percent cell proliferation that shows that anantibody against an epitope of the MUC1 receptor which is proximal tothe cell surface, and that dimerizes the receptor, enhances cellproliferation in a manner typical of a growth factor/receptor-antibodyinteraction;

[0039]FIG. 5 is a graph of percent cell proliferation that shows that anantibody against an epitope of the MUC1 receptor which is proximal tothe cell surface, and that dimerizes the receptor, dramatically enhancescell proliferation;

[0040]FIG. 6 (color) is an image of a section of a 96-well plateillustrating a color change assay in which a ligand(s) present in thelysates of cells that express MUC1, binds to and dimerize the His-PSMGFRpeptide, derived from MUC1, which is immobilized on gold colloids, whilelysates from cells that do not express MUC1 do not;

[0041]FIG. 7 (color) is an image of 96-well plate illustrating acolloid-based color-change binding assay between a MUC1-derived peptideand a ligand(s) present in a crude cell lysate; addition of imidazole,which releases the probe peptide from the colloid, causes a reversal ofthe color change, which argues that the color change is the result of aspecific interaction rather than random colloid aggregation;

[0042] FIGS. 8A-D (color) shows a colloid-based color change assay in96-well plates in which a ligand present in a cell lysate causeddimerization of a MUC1-derived peptide and that the degree of colorchange, which indicates an amount of ligand present, was a function ofwhich cell line supplied the lysate;

[0043]FIG. 9 (color) is a silver-stained gel showing ligands that werefished out of cell lysates using the PSMGFR peptide, in the presence ofthe protease inhibitor PMSF;

[0044]FIG. 10 (color) is a silver-stained gel showing ligands that werefished out of cell lysates using the PSMGFR peptide, in the absence ofthe protease inhibitor PMSF;

[0045]FIG. 11 (color) is an image of 96-well plate illustrating acolor-change binding assay between a MUC1-derived peptide and aligand(s) present in a crude cell lysate from cells that overexpressMUC1;

[0046]FIG. 12 (color) is an image of a 96-well plate illustrating acolor-change drug-screening assay used to detect inhibitors of theMUC1-Ligand interaction;

[0047]FIG. 13 shows a histogram illustrating the selective inhibition ofproliferation of tumor cells that aberrantly express the MUC1 receptor,in response to treatment with compounds of the invention, and lack of aneffect on cells that do not express MUC1;

[0048]FIG. 14 (color) is an image of a 96-well plate illustrating acolor-change drug-screening assay identifying several compounds thatintefere with the interaction of the MGFR portion of the MUC1 receptorand a multimerizing ligand(s);

[0049]FIG. 15 shows a histogram illustrating the selective inhibition ofproliferation of tumor cells that aberrantly express the MUC1 receptor,in response to treatment with drugs that specifically inhibit MUC1positive cells;

[0050]FIG. 16 shows a histogram illustrating the nonselective inhibitionof proliferation of cells in response to treatment with drugs thatnon-specifically inhibit cell proliferation;

[0051]FIG. 17 shows a histogram illustrating that drugs that selectivlyinhibit proliferation of tumor cells that aberrantly express the MUC1receptor bind to the PSMGFR, while drugs that non-selectively inhibitcell proliferation do not;

[0052]FIG. 18 is a graph showing that the inhibition of MUC1-dependentcell proliferation induced by an anti-tumor drug identified inaccordance with the invention, is modulated when a synthetic peptide,corresponding to the portion of MUC1 that remains at the cell followingcleavage, competitively inhibits the drug-cell surface receptorinteraction;

[0053]FIG. 19 (color) is a comassie blue-stained gel showing that thePSMGFR peptide runs at an apparently higher molecular weight afterincubation with cell;

[0054]FIG. 20 (color) is an image of a 96-well plate illustrating acolor-change ligand binding assay illustrating that inhibiting enzymaticmodification of PSMGFR prevents it binding to ligands.

DETAILED DESCRIPTION OF THE INVENTION

[0055] Definitions:

[0056] The term “MUC1 Growth Factor Receptor” (MGFR) is a functionaldefinition meaning that portion of the MUC1 receptor that interacts withan activating ligand, such as a growth factor, to promote cellproliferation. The MGFR region of MUC1 is that portion that is closestto the cell surface and is defined by most or all of the PSMGFR. TheMGFR is inclusive of both unmodified peptides and peptides that haveundergone enzyme modifications, such as, for example, phosphorylation,glycosylation, etc. Results of the invention are consistent with amechanism in which this portion is made accessible to the ligand uponMUC1 cleavage at a site associated with tumorigenesis that causesrelease of the IBR from the cell.

[0057] The term “Interchain Binding Region” (IBR) is a functionaldefinition meaning that portion of the MUC1 receptor that binds stronglyto identical regions of other MUC1 molecules giving MUC1 the ability toaggregate (i.e. self-aggregate) with other MUC1 receptors via the IBRsof the respective receptors. This self-aggregation may contribute toMUC1 receptor clustering, observed in healthy cells.

[0058] In a preferred embodiment, the IBR may be approximately definedas a stretch of at least 12 to 18 amino acid sequence within the regionof the human MUC1 receptor defined as comprising amino acids 507 to 549of the extracellular sequence of the MUC1 receptor, with amino acids 525through 540 and 525 through 549 especially preferred (numbers refer toAndrew Spicer et al., J. Biol. Chem Vol 266 No. 23, 1991 pgs.15099-15109; these amino acid numbers correspond to numbers 1067, 1109,1085, 1100, 1085, 1109 of Genbank accession number P15941; PID G547937,SEQ ID NO: 10) or fragments, functional variants or conservativesubstitutions thereof.

[0059] The term “cleaved IBR” means the IBR (or a portion thereof) thathas been released from the receptor molecule segment which remainsattached to the cell surface. The release may be due to enzymatic orother cleavage of the IBR. As used herein, when the IBR is “at thesurface of a cell”, it means the IBR is attached to the portion of thecell surface receptor that has not been shed, or cleaved. The cleavedIBR of interest is a “disease-associated cleavage”, i.e. that type ofcleavage that can result in cancer.

[0060] The term “Constant Region” (CR) is any non-repeating sequence ofMUC1 that exists in a 1:1 ratio with the IBR and forms part of theportion of MUC1 that is shed upon cleavage in healthy and tumorigenesiccells.

[0061] The term “Repeats” is given its normal meaning in the art.

[0062] The term “Primary Sequence of the MUC1 Growth Factor Receptor”(PSMGFR) is a peptide squence, defined below (See Table 1—SEQ ID NO: 7),that defines most or all of the MGFR. The PSMGFR is inclusive of bothunmodified peptides and peptides that have undergone enzymemodifications, such as, for example, phosphorylation, glycosylation,etc. The histidine-tagged PSMGFR (See Table 1—SEQ ID NO: 2) isabbreviated herein as His-PSMGFR.

[0063] The term “Extended Sequence of the MUC1 Growth Factor Receptor”(ESMGFR) is a peptide squence, defined below (See Table 1—SEQ ID NO: 3),that defines all of His-PSMGFR plus 9 amino acids of the proximal end ofPSIBR.

[0064] PSIBR is a peptide squence, defined below (See Table 1—SEQ ID NO:8), that defines most or all of the IBR.

[0065] The term “separation” means physical separation from a cell, i.e.a situation in which a portion of MUC 1 that was immobilized withrespect to a cell is no longer immobilized with respect to that cell.E.g. in the case of cleavage of a portion of MUC 1, the portion that iscleaved is “separated” if it is free to migrate away from the cell andthereafter may be detected in a bodily fluid, or immobilized at alocation remote from the cell from which it was cleaved such as anothercell, a lymph node, etc.

[0066] The term “binding” refers to the interaction between acorresponding pair of molecules that exhibit mutual affinity or bindingcapacity, typically specific or non-specific binding or interaction,including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc.

[0067] The term “binding partner” refers to a molecule that can undergobinding with a particular molecule. Biological binding partners areexamples. For example, Protein A is a binding partner of the biologicalmolecule IgG, and vice versa.

[0068] The term “aggregate” (noun) means a plurality of cell surfacereceptors or fragments thereof (e.g. MUC 1) immobilized with respect toeach other with or without an intermediate auxialliary to the hostsystem. This includes self-aggregation of healthy receptors at a cellsurface; self-aggregation of cleaved receptors or fragments bound toeach other; cleaved receptors or fragments bound to receptors orfragments attached to a cell surface; receptors or fragments, whetherattached to a cell or cleaved, immobilized with respect to each othervia an intermediate auxialliary to the host. “Intermediate auxialliaryto the host system” includes a synthetic species such as a polymer,dendrimer, etc., or a naturally-occurring species, for example an IgMantibody, which is not simply naturally present in the host system butis added to the host system from a source external to the host system.This excludes aggregation that is the result of an intermediatenaturally present in the host system such as a growth factor that cancause disease-associated aggregation (“Inductive multimerization”).“Aggregate” (verb) or “aggregation” means the process of forming anaggregate (noun).

[0069] “Inductive multimerization” refers to aggregation wherein theaggregate formed can act to induce the cells to grow or proliferate.Inductive multimerization typically involves dimerization ortetramerization of cell surface receptors, for example by a growthfactor or other activating ligand, but can also involve higher ordermultimerization, so long as the degree of multimerization is not sogreat as to mimic natural receptor clustering, in a particular celltype, which prevents receptors from signalling the cell to grow orproliferate.

[0070] “Preventative clustering” refers to multimerization of receptorsto form an aggregate involving a sufficient number of receptors to mimicnatural receptor clustering, in a particular cell type, which preventsreceptors from signalling the cell to grow or proliferate, for examplewith an intermediate auxialliary to the host system.

[0071] A “ligand” to a cell surface receptor, refers to any substancethat can interact with the receptor to temporarily or permanantly alterits structure and/or function. Examples include, but are not limited tobinding partners of the receptor and agents able to alter the chemicalstructure of the receptor (e.g. modifying enzymes).

[0072] An “activating ligand” refers to a ligand able to effectinductive multimerization of cell surface receptors. Activating ligandscan include, but are not limited to, a single molecular species withgreater than one active site able to bind to a receptor; a dimer, atetramer, a higher multimer, or a complex comprising a plurality ofmolecular species. In the context of MUC1 tumor cells, an activatingligand can be a species produced by the cells that interacts with theMGFRs on the surface of the MUC1 tumor cells in a manner that effectsinductive multimerization.

[0073] A “growth factor” refers to a species that may or may not fallinto a class of previously-identified growth factors, but which acts asa growth factor in that it acts as an activating ligand.

[0074] A “MUC1 presenting cell” refers to both non-cancerous andcancerous cells expressing MUC1 and/or MGFRs on the surface. A “MUC1tumor cell” or “MUC1 cancer cell” or “cancerous MUC1 cell” refers to acancerous tumor cell that aberrantly expresses MUC1 and/or MGFR on itssurface.

[0075] “Colloids”, as used herein, means nanoparticles, i.e. very small,self-suspendable or fluid-suspendable particles including those made ofmaterial that is, e.g., inorganic or organic, polymeric, ceramic,semiconductor, metallic (e.g. gold), non-metallic, crystalline,amorphous, or a combination. Typically, colloid particles used inaccordance with the invention are of less than 250 nm cross section inany dimension, more typically less than 100 nm cross section in anydimension, and in most cases are of about 2-30 nm cross section. Oneclass of colloids suitable for use in the invention is 10-30 nm in crosssection, and another about 2-10 nm in cross section. As used herein thisterm includes the definition commonly used in the field of biochemistry.

[0076] As used herein, a component that is “immobilized relative to”another component either is fastened to the other component or isindirectly fastened to the other component, e.g., by being fastened to athird component to which the other component also is fastened, orotherwise is transitionally associated with the other component. Forexample, a signaling entity is immobilized with respect to a bindingspecies if the signaling entity is fastened to the binding species, isfastened to a colloid particle to which the binding species is fastened,is fastened to a dendrimer or polymer to which the binding species isfastened, etc. A colloid particle is immobilized relative to anothercolloid particle if a species fastened to the surface of the firstcolloid particle attaches to an entity, and a species on the surface ofthe second colloid particle attaches to the same entity, where theentity can be a single entity, a complex entity of multiple species, acell, another particle, etc.

[0077] “Signaling entity” means an entity that is capable of indicatingits existence in a particular sample or at a particular location.Signaling entities of the invention can be those that are identifiableby the unaided human eye, those that may be invisible in isolation butmay be detectable by the unaided human eye if in sufficient quantity(e.g., colloid particles), entities that absorb or emit electromagneticradiation at a level or within a wavelength range such that they can bereadily detected visibly (unaided or with a microscope including anelectron microscope or the like), or spectroscopically, entities thatcan be detected electronically or electrochemically, such asredox-active molecules exhibiting a characteristic oxidation/reductionpattern upon exposure to appropriate activation energy (“electronicsignaling entities”), or the like. Examples include dyes, pigments,electroactive molecules such as redox-active molecules, fluorescentmoieties (including, by definition, phosphorescent moieties),up-regulating phosphors, chemiluminescent entities,electrochemiluminescent entities, or enzyme-linked signaling moietiesincluding horseradish peroxidase and alkaline phosphatase. “Precursorsof signaling entities” are entities that by themselves may not havesignaling capability but, upon chemical, electrochemical, electrical,magnetic, or physical interaction with another species, become signalingentities. An example includes a chromophore having the ability to emitradiation within a particular, detectable wavelength only upon chemicalinteraction with another molecule. Precursors of signaling entities aredistinguishable from, but are included within the definition of,“signaling entities” as used herein.

[0078] As used herein, “fastened to or adapted to be fastened”, in thecontext of a species relative to another species or to a surface of anarticle, means that the species is chemically or biochemically linkedvia covalent attachment, attachment via specific biological binding(e.g., biotin/streptavidin), coordinative bonding such as chelate/metalbinding, or the like. For example, “fastened” in this context includesmultiple chemical linkages, multiple chemical/biological linkages, etc.,including, but not limited to, a binding species such as a peptidesynthesized on a polystyrene bead, a binding species specificallybiologically coupled to an antibody which is bound to a protein such asprotein A, which is attached to a bead, a binding species that forms apart (via genetic engineering) of a molecule such as GST or Phage, whichin turn is specifically biologically bound to a binding partnercovalently fastened to a surface (e.g., glutathione in the case of GST),etc. As another example, a moiety covalently linked to a thiol isadapted to be fastened to a gold surface since thiols bind goldcovalently. Similarly, a species carrying a metal binding tag is adaptedto be fastened to a surface that carries a molecule covalently attachedto the surface (such as thiol/gold binding) which molecule also presentsa chelate coordinating a metal. A species also is adapted to be fastenedto a surface if a surface carries a particular nucleotide sequence, andthe species includes a complementary nucleotide sequence.

[0079] “Covalently fastened” means fastened via nothing other than oneor more covalent bonds. E.g. a species that is covalently coupled, viaEDC/NHS chemistry, to a carboxylate-presenting alkyl thiol which is inturn fastened to a gold surface, is covalently fastened to that surface.

[0080] “Specifically fastened” or “adapted to be specifically fastened”means a species is chemically or biochemically linked to anotherspecimen or to a surface as described above with respect to thedefinition of “fastened to or adapted to be fastened”, but excluding allnon-specific binding.

[0081] Certain embodiments of the invention make use of self-assembledmonolayers (SAMs) on surfaces, such as surfaces of colloid particles,and articles such as colloid particles having surfaces coated with SAMs.In one set of preferred embodiments, SAMs formed completely of syntheticmolecules completely cover a surface or a region of a surface, e.g.completely cover the surface of a colloid particle. “Syntheticmolecule”, in this context, means a molecule that is not naturallyoccurring, rather, one synthesized under the direction of human orhuman-created or human-directed control. “Completely cover” in thiscontext, means that there is no portion of the surface or region thatdirectly contacts a protein, antibody, or other species that preventscomplete, direct coverage with the SAM. I.e. in preferred embodimentsthe surface or region includes, across its entirety, a SAM consistingcompletely of non-naturally-occurring molecules (i.e. syntheticmolecules). The SAM can be made up completely of SAM-forming speciesthat form close-packed SAMs at surfaces, or these species in combinationwith molecular wires or other species able to promote electroniccommunication through the SAM (including defect-promoting species ableto participate in a SAM), or other species able to participate in a SAM,and any combination of these. Preferably, all of the species thatparticipate in the SAM include a functionality that binds, optionallycovalently, to the surface, such as a thiol which will bind to a goldsurface covalently. A self-assembled monolayer on a surface, inaccordance with the invention, can be comprised of a mixture of species(e.g. thiol species when gold is the surface) that can present (expose)essentially any chemical or biological functionality. For example, theycan include tri-ethylene glycol-terminated species (e.g. tri-ethyleneglycol-terminated thiols) to resist non-specific adsorption, and otherspecies (e.g. thiols) terminating in a binding partner of an affinitytag, e.g. terminating in a chelate that can coordinate a metal such asnitrilotriacetic acid which, when in complex with nickel atoms, capturesa metal binding tagged-species such as a histidine-tagged bindingspecies. The present invention provides a method for rigorouslycontrolling the concentration of essentially any chemical or biologicalspecies presented on a colloid surface or any other surface. Withoutthis rigorous control over peptide density on each colloid particle,co-immobilized peptides would readily aggregate with each other to formmicro-hydrophobic-domains that would catalyze colloid-colloidaggregation in the absence of aggregate-forming species present in asample. This is an advantage of the present invention, over existingcolloid agglutination assays. In many embodiments of the invention theself-assembled monolayer is formed on gold colloid particles.

[0082] The kits described herein, contain one or more containers, whichcan contain compounds such as the species, signaling entities,biomolecules, and/or particles as described. The kits also may containinstructions for mixing, diluting, and/or administrating the compounds.The kits also can include other containers with one or more solvents,surfactants, preservative and/or diluents (e.g. normal saline (0.9%NaCl, or 5% dextrose) as well as containers for mixing, diluting oradministering the components to the sample or to the patient in need ofsuch treatment.

[0083] The compounds in the kit may be provided as liquid solutions oras dried powders. When the compound provided is a dry powder, the powdermay be reconstituted by the addition of a suitable solvent, which alsomay be provided. Liquid forms of the compounds may be concentrated orready to use. The solvent will depend on the compound and the mode ofuse or administration. Suitable solvents for are well known for drugcompounds and are available in the literature.

[0084] The term “cancer”, as used herein, may include but is not limitedto: biliary tract cancer; bladder cancer; brain cancer includingglioblastomas and medulloblastomas; breast cancer; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer;gastric cancer; hematological neoplasms including acute lymphocytic andmyelogenous leukemia; multiple myeloma; AIDS-associated leukemias andadult T-cell leukemia lymphoma; intraepithelial neoplasms includingBowen's disease and Paget's disease; liver cancer; lung cancer;lymphomas including Hodgkin's disease and lymphocytic lymphomas;neuroblastomas; oral cancer including squamous cell carcinoma; ovariancancer including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreatic cancer; prostate cancer;rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer includingmelanoma, Kaposi's sarcoma, basocellular cancer, and squamous cellcancer; testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullar carcinoma; and renal cancer including adenocarcinoma and Wilmstumor. Preferred cancers are; breast, prostate, lung, ovarian,colorectal, and brain cancer.

[0085] The term “cancer treatment” as described herein, may include butis not limited to: chemotherapy, radiotherapy, adjuvant therapy, or anycombination of the aforementioned methods. Aspects of treatment that mayvary include, but are not limited to: dosages, timing of administration,or duration or therapy; and may or may not be combined with othertreatments, which may also vary in dosage, timing, or duration. Anothertreatment for cancer is surgery, which can be utilized either alone orin combination with any of the aforementioned treatment methods. One ofordinary skill in the medical arts may determine an appropriatetreatment.

[0086] An “agent for prevention of cancer or tumorigenesis” means anyagent that counteracts any process associated with cancer ortumorigenesis described herein. For example, an agent that interactswith (e.g. binds to) to MGFR thereby reducing or preventing interaction,with MGFR, of an agent that promotes tumorigenesis by its interactionwith MGFR.

[0087] An “agent that reduces cleavage of a cell surface receptorinterchain binding region” as used herein is any composition thatprevents or reduces cleavage of the MUC1 receptor between the MGFR andthe IBR that would otherwise occur in the absence of the agent. Cleavageof the receptor between the MGFR and the IBR can be caused by activityof enzymes that are membrane-associated or soluble. Some of theseenzymes are directly responsible for cleavage. Other enzymes can affectcleavage, (e.g. prevent cleavage at a particular location) by modifyingMUC1 with sugar groups or phosphates that mask a recognition epitopeassociated with cleavage. Other enzymes can promote cleavage at aparticular location by modifying MUC1 with sugar groups or phosphatesthat create a recognition motif for cleavage at that location. One wayto select agents that reduce cleavage of a cell surface receptor IBR isto first identify enzymes that affect cleavage as described above, andscreen agents, and their analogs, for their ability to alter theactivity of those enzymes. Another way is to test agents that are knownto affect the activity of similar enzymes (e.g. from the same family)for their ability to alter the site of cleavage of MUC1, and tosimilarly test analogs of these agents. Alternatively, agents arescreened in a cell-free assay containing the enzyme and MUC1 receptors,and the the rate or position of cleavage measured by antibody probing,Polymerase Chain Reaction (PCR), or the like. Alternatively, withoutfirst identifying enzymes that affect MUC1, agents are screened againstcells that present MUC1 for the agents' ability to alter cleavage siteor the rate of cleavage of MUC1. For example, agents can be screened inan assay containing whole cells that present MUC1 and aggregationpotential of the cell supernatant can be measured, an indication of theamount of IBR that remains attached to the cleaved portion of MUC1 ,i.e. the degree of cleavage between MGFR and IBR. In another technique,agents can be screened in an assay containing whole cells that presentMUC1, the supernatant removed, and the cell remain tested foraccessibility of the MGFR portion, e.g. using a labeled antibody to theMGFR. Agents can be identified from commercially available sources suchas molecular libraries, or rationally designed based on known agentshaving the same functional capacity and tested for activity using thescreening assays.

[0088] An “agent that reduces cleavage of the MUC1 receptor” is anycomposition that prevents or reduces cleavage of the MUC1 receptor atany location. Such an agent can be used to treat a subject having canceror at risk for developing cancer because if cleavage is prevented, thenthe accessibility of the MGFR, a functional receptor associated withcancer, is reduced or prevented. Such agents can be selected by exposingcells to a candidate agent and determine, in the supernatant, the amountof cleaved MUC1 receptor, relative to a control.

[0089] A subject, as used herein, refers to any mammal (preferably, ahuman), and preferably a mammal that may be susceptible to tumorigenesisor cancer associated with the abherrant expression of MUC1. Examplesinclude a human, non-human primate, cow, horse, pig, sheep, goat, dog,or cat. Generally, the invention is directed toward use with humans.

[0090] The samples used herein are any body tissue or body fluid sampleobtained from a subject. Preferred are body fluids, for example lymph,saliva, blood, urine, and the like. Blood is most preferred. Samples oftissue and/or cells for use in the various methods described herein canbe obtained through standard methods including, but not limited to:tissue biopsy, including punch biopsy and cell scraping, needle biopsy,and collection of blood or other bodily fluids by aspiration or othermethods.

[0091] The following patent applications and publications areincorporated herein by reference: international patent applicationserial no. PCT/US00/01997, filed Jan. 25, 2000, entitled “Rapid andSensitive Detection of Aberrant Protein Aggregation in NeurodegenerativeDiseases”, published as no. WO 00/43791, international patentapplication serial no. PCT/US00/01504, filed Jan. 21, 2000, entitled“Assays involving Colloids and Non-Colloidal Structures”, published Jul.27, 2000 as international patent publication no. WO 00/34783, U.S.patent application Ser. No. 09/631,818, filed Aug. 3, 2000, entitled“Rapid and Sensitive Detection of Protein Aggregation”, a U.S.provisional patent application by Bamdad, et al., serial No. 60/248,865,filed Nov. 15, 2000, entitled “Endostatin-Like Angiogenesis Inhibition,”and a U.S. Utility Application Application of same title filed Nov. 15,2001.

[0092] The present invention involves, generally, novel moleculartargets for drug screening, therapeutics and diagnostics related tocancers that are characterized by the aberrant expression of a class ofcell surface receptors characterized by interchain binding regions. Onesuch set of cancers are those characterized by the aberrant expressionof MUC1. Much of the description of the invention herein involves cellsthat aberrantly express MUC1. It is to be understood that in theseinstances the description is to be considered exemplary, and that theprinciples of the invention apply to other cell surface receptors thatfunction by a similar mechanism. With the disclosure herein, those ofordinary skill in the art will readily be able to identify other cellsurface receptors that function by this or a similar mechanism, and toapply the invention to those cancers characterized by aberrantexpression of receptors. The invention is based on a novel mechanisminvolving cell surface receptors that have regions that self-aggregate,exemplified by MUC1, which was elucidated by the inventors.

[0093] MUC1 comprises several regions termed herein as follows, recitedin an order starting from the region closest to the cell surface andprogressing away from the cell. In at least one U.S. provisional patentapplication (“earlier application(s)”) filed by the same inventors,entitled “Tumor Markers and Drug Screening for TumorogenesisInhibition”, relating to MUC1 diagnostics and other techniques, at leastone region of MUC1 was defined differently. It is to be understood thatthe following definition supercedes. Those of ordinary skill in the artwill understand the invention in all its aspects from the description ofportions of MUC1 referred to differently in the earlier application(s)and in the current application, and the relation of the earlierapplication(s) to this application. (1) the PSMGFR was referred to inthe earlier application(s) as an FLR region or peptide); (2) the PSIBRwas referred to in the earlier application(s) as a CM region or peptide.The basic structure of the MUC1 receptor is illustrated in FIG. 1. Thereceptor, as illustrated comprises: 1) cytoplasmic tail; 2)transmembrane section; 3) MGFR; 4) IBR, and 5) repeats.

[0094] One aspect of the present invention features the discovery that aspecific region of the MUC1 receptor, i.e., the IBR, binds strongly toidentical regions of other MUC1 molecules. That is, the MUC1 receptorhas the ability to aggregate (i.e. self-aggregate) with other MUC1receptors via the IBR of the respective receptors. This self-aggregationmay contribute to MUC1 receptor clustering, observed in healthy cells.The discovery that the IBR portion of the MUC1 receptor self-aggregatesis consistent with the following mechanistic model for which theinventors present supporting evidence. Mechanistic model: (1) receptorclustering is associated with the healthy state because the aggregatedIBR portions block access of ligands, such as growth factors, modifyingenzymes and the like to the neighboring extracellular portions of theMUC1 receptor that act as the functional receptor; clustering alsoblocks access of intracellular tails to intracellular modifying enzymesand signaling ligands; (2) when the MUC1 receptor is cleaved at aposition that releases the IBR, the critical force that keeps thereceptors clustered is lost and receptors are free to migrate within thecell membrane or interact with modifying enzymes, secreted ligands suchas activating ligands or growth factors or other cell surface receptors;these interactions could involve a new, inductive multimerization state,such as dimerization, that triggers a cell proliferation signalingcascade.

[0095] Cleavage of MUC1 may occur at a site at or near the C-terminalboundary of the IBR in tumor or cancer cells (between the cell and theIBR), releasing the IBR from the cell. Alternatively, cleavage of MUC1may occur within the IBR itself to cause sufficient disrupting of theIBRs such that the ability to self-aggregate is lost with the resultthat the MGFR becomes accessible to agents or ligands. As described inExample 1b, the addition of 9 amino acids of the IBR region to a peptidefrom the MGFR region (which does not self-aggregate), confers someability to self-aggregate. Alternatively, loss of aggregation of MUC1receptors need not necessarily be the result of cleavage. For example,an IBR can be absent as a result of alternative splicing of the MUC1gene.

[0096] Before the present invention, a ligand(s) for the MUC1 receptorhad not been conclusively identified. Research articles suggested thatthe shed portion of the MUC1 receptor becomes a ligand for the portionof the receptor that remains attached to the cell surface aftercleavage. In the present invention, this hypothesis was tested, and itwas determined not to be the case. Further, it was determined thataltered sites of cleavage of the MUC1 receptor could result in alteredfunction. In a calorimetric colloid binding assay (described inPCT/US00/01997, referenced above, as well as some other assays of theabove-incorporated patent applications/publications) various fragmentsof the MUC1 receptor were tested for their ability to interact with eachother fragment of MUC1 as well as for their ability to self-aggregate.We found that one portion of the receptor, fairly close to the cellsurface, aggregated with itself in a high affinity interaction. Thissuggested that this portion of the MUC1 receptor, which we termed theinterchain binding region, kept the receptors tightly clustered in ahealthy cell, and that enzyme cleavage of MUC1 at a site that releasedthe IBR, would cause the receptors not remain clustered. This clusteringmay affect cell signaling in two ways. First, the clustering of thereceptors on the cell surface may restrict access to portions of thereceptor that are binding sites for ligands such as modifying enzymes orgrowth factors. Secondly, as is appreciated by those skilled in the art,the intracellular portions (cytoplasmic tails) of many cell surfacereceptors are involved in signaling cascades that control programmedcell growth (proliferation) as well as programmed cell death(apoptosis). Receptors that are tightly clustered on the cell surfacealso have clustered cytoplasmic tails within the cell, which may preventthem from interacting with intracellular proteins involved inintracellular signaling.

[0097] In some cases, the MUC1 receptor may be cleaved to release theIBR, from the cell surface. Alternatively, cleavage can result in arelease of a sufficient portion of the IBR that causes the MUC1 receptorto lose the ability to self-aggregate. Loss of aggregation of MUC1 mayhave several ramifications. Release of the IBR or sufficient portion ofthe IBR from the cell surface allows the receptors to evenly distributeon the cell surface, leaving the cytoplasmic tails free to associatewith intracellular signaling proteins. External agents, such asmodifying enzymes and/or activating ligands, are then able to bind tothe remaining extracellular portion of the receptor and inducedisease-associated signals, either via a change in the multimerizationstate, i.e., inductive multimerization, or as an induced conformationalchange. As is appreciated by those of ordinary skill in the art, ligandssuch as growth factors and hormones often induce receptor dimerizationwhich triggers, in turn, an intracellular signaling cascade.

[0098] Cell proliferation may result from accessibility of the MGFRportion to an activating ligand which can interact with the MGFRportion. For example, the self-aggregating IBR of the MUC1 receptor mayform a dense reticulum which sterically prevents a ligand such as agrowth factor from intreacting with the MGFR portion of the receptor,which is proximal to the cell relative to the IBR. In a cancerous ortumor cell, this reticulum may be lost, allowing ligand interaction withthe MGFR.

[0099] The above mechanistic model is consistent with a mechanismwhereby the portion of the MUC1 receptor, that remains attached to thecell surface after shedding of the IBR region, i.e. the MGFR, functionsas a receptor for ligands that trigger cell proliferation. Evidence isalso presented herein that indicates that this portion of the receptoris enzyme modified before it is able to be recognized by at least one ofits ligands (See Example 8). This mechanism is demonstrated herein witha showing that: (a) an interaction between a ligand and this portion ofthe MUC1 receptor (MGFR), which dimerizes the receptor, triggers cellproliferation; and (b) blocking the interaction of this portion of theMUC1 receptor (MGFR) with its ligand(s), blocks cell proliferation. Whentumor cell lines, in which the MUC1 receptor is homogeneously expressedacross the entire cell surface, are treated with an IgG antibody raisedagainst the MGFR portion of the MUC1 receptor, the rate of cellproliferation is greatly enhanced, see FIG. 5. Since IgG antibodies arebivalent, i.e. one antibody simultaneously binds to two adjacent MGFRportions on the cell surface, these results demonstrate that theantibody acts as an activating ligand, mimicing the effect of a growthfactor, which dimerizes MGFR portions, and thus triggers a cellproliferation signaling cascade which is consistent with signaling viathe cytoplasmic tails of the receptors. This finding leads to twoconclusions. First, an activating ligand(s) that binds to the MGFRportion of the MUC1 receptor causes inductive multimerization of thereceptor. Secondly, an effective therapeutic strategy is therefore toblock the MGFR portion of the receptor with a monomeric composition,thus preventing inductive multimerization and subsequent signalingcascades. For example, a single chain, or monovalent, antibody raisedagainst the MGFR portion of the MUC1 receptor would function as aneffective anti-cancer therapeutic. Another therapeutic strategy is toblock the activity of enzymes that modify the receptor, which may berequired for some ligand binding

[0100] The inventors have also discovered that cells that overexpressthe MUC1 receptor also have increased levels of a ligand(s) thatdimerizes the MGFR present in the lysates and supernatants of thesecells, see Example 3b and FIGS. 8A-D for details. In the colloid-colloidinteraction experiment, described in Example 3b, ligands thatsimultaneously bind more than one colloid-immobilized receptor, i.e.dimerize, cause a solution color change from pink to blue. Gold colloidsthat presented synthetic peptides derived from the MGFR portion of theMUC1 receptor (His-PSMGFR) were incubated with lysates/supematants fromvarious cells lines known to overexpress, express, or not express theMUC1 receptor. Lysates from HTB-133 (T47D) cells, which overexpressesMUC1 (see Table 2), caused colloid suspensions to turn blue within 15minutes, indicating a high concentration of a ligand(s) in the lysatethat interacts with the colloid-immobilized MGFR-derived peptides. Inexperiments with lysates from other cell lines, the rate of color changeof the colloid solution, which indicates the amount of ligand present,correlated to the degree of expression of the MUC1 receptor in thosecell lines with cells.

[0101] More than one species may be a physiologically relevant ligandfor this portion of the MUC1 receptor. Enzymes that modify the receptormay be relevant ligands of this portion of the receptor. For example,one ligand may bind monomerically to an unmodified MGFR portion of theMUC1 receptor, while another ligand, with a different function, such asinductive multimerization, may recognize an enzyme-modified version ofthe receptor. Because the experiment described above, Example 3b, wasperformed in cell lysate/supernatants, it is important to note thatseveral receptor-ligand interactions, including enzymatic modificationsto the receptor, may be taking place, wherein only the ligand(s)dimerization (or multimerization) of the MGFR portion, results in asolution color change. In an experiment similar to Example 3b, (Example8) the enzyme inhibitor, PMSF was added to the lysate prior to theintroduction of the colloids bearing the synthetic peptide His-PSMGFR,see Table 1 SEQ ID # 2. Referring now to FIG. 20, solutions thatcontained PMSF did not undergo the solution color change. This result isconsistent with a mechanism in which the MGFR portion of the MUC1receptor is first enzyme mofdified before it is recognized by theligand(s) that dimerize or multimerize the receptor.

[0102] The inventors reasoned that prior attempts by others to identifyligands for the MUC1 receptor were hampered by the self-aggregationproperties of the receptor. Therefore, only the MGFR portion of thereceptor was used as bait to fish ligands out of lysates andsupernatants. The His-PSMGFR sequence of Table 1, was immobilized onNTA-nickel beads via the histidine tag of the peptide, the beads werethen incubated with lysates and supernatants from a variety of celltypes, including cancer cell lines that overexpress MUC1. Enzymeinhibitors such as PMSF were added to some of the lysates andsupernatants to circumvent problems of alternative ligands binding tomodified versions of the peptide. Following incubation of the cellsupernatants with the PSMGFR-presenting beads, the beads were washed,then the peptide-ligand complexes eluted from the NTA-Nickel beads byadding excess imidazole. Captured ligands from the probe peptides,eluates, were separated using standard SDS-PAGE methods. Protein bandswere excised from the gels and analyzed to identify the target ligands.Standard methods for protein analysis including peptide micro sequencingand tandem mass spec were used in these studies. Other methods can beused to identify MUC1 ligands, including ligand fishing with beads,MALDI mass spec, and the like.

[0103] Accordingly, another aspect of the invention involves theidentification of ligands, derived from lysates from a cell lineselected from the group consisting of HTB-133, CRL-1504, and CRL-1500,that bind to the MGFR portion of the MUC1 cell surface receptor, seeFIGS. 9 and 10. In some embodiments, the ligands may include sequence(s)from: Metastasis Inhibition Factor NM23, 14-3-3, Cathepsin D, annexin,Beta lipotropin (Beta-LPH), beta-melanotropin or Beta-MSH. In otherembodiments, the biomolecule that binds to the MGFR portion may be acleavage product of proopiomelanocortin (POMC). In all embodiments, thepreferred cell surface receptor is MUC1. In one embodiment, the MGFRportion includes some or all of the sequence from the PSMGFR peptide(SEQ ID NO: 7). These ligands may exist in a multimeric state includingdimers, tetramers, or complexes containing some or all of these ligandspecies. In one aspect, the invention involves modification and use ofthe above species as anti-cancer agents.

[0104] In one embodiment the ligand is a 23 kD protein. In anotherembodiment, the ligand is approximately 17 kD. In a preferredembodiment, the ligand identified is a protein that migrates through agel with an apparent molecular weight of 35 kD. Since this species ismuch more apparent when the protease inhibitor PMSF is not added to thelysate (FIG. 10), this protein may be an enzyme that modifies the MUC1receptor or a ligand that recognizes the unmodified version of thereceptor. Experiments were performed as described in more detail belowin Example 4b to characterize these species.

[0105] Peptide sequences contained within both the 17 kD and the 23 kDbands (PMSF added to lysate) corresponded to a protein known asMetastasis Inhibition Factor NM23, which has been implicated in both thepromotion and inhibition of metastasis of human cancers. Whether therole of NM23 is a tumor supressor or promoter may depend on the type ofcancer. In ovarian, colon and neuroblastoma tumors, NM23 overexpressionhas been linked to a more malignant phenotype (Schneider J, Romero H,Ruiz R, Centeno M M, Rodriguez-Escudero F J, “NM23 expression inadvanced and borderline ovarian carcinoma”, Anticancer Res, 1996;16(3A): 1197-202). However, breast cancer studies indicate that reducedexpression of NM23 correlates with poor prognosis (Mao H, Liu H, Fu X,Fang Z, Abrams J, Worsham M J, “Loss of NM23 expression predicts distalmetastases and poorer survival for breast cancer”, Int J Oncol 2001March;18(3):587-91). Because NM23 exists as a hexamer, inMUC1-presenting cells, it may function to hold MUC1 receptors in aclustered configuration to restrict access of the MGFR to modifying andactivating ligands. The sequences that were identified from the proteingel band described in FIGS. 9 and 10 and that also exist in MetastasisInhibition Factor NM23 are shown below in Table 4

[0106] Peptide sequences that are associated with the 35 kD gel band(PMSF NOT added to lysate) corresponded to more than one proteinspecies, including 14-3-3, which is a signaling protein implicated inmany cancers, and cathepsin D, which is a protease and is alsoimplicated in tumor progression. 14-3-3 exists as a dimer and cansimultaneously bind to two, identical phospho-serine peptides. Thisprotein has been shown to be involved in intracellular signaling, butparticularly relevant to this invention is the fact that 14-3-3 has beenshown to be secreted by some cell types, including dendritic cells.Ligands that bind to the MGFR portion of the receptor to induceinductive multimerization would tend to be secreted factors. Thisprotein would dimerize the MUC1 receptor to trigger cell proliferation,which is consistent with the mechanism presented herein. Cathepsin D isa protease and may be involved in the cleavage of the MUC1 receptor.

[0107] Yet another aspect of the invention involves the identificationof other ligands, also derived from supernatants from a cell lineselected from the group consisting of HTB-133, CRL-1504, and CRL-1500,that bind to the MGFR portion of the MUC1 cell surface receptor. In oneembodiment, the ligand identified is a protein that migrates through agel with an apparent molecular weight of 55 kD. In another embodiment,the ligand is approximately 70 kD; 80 kD; or 100 kD. In a particularembodiment, the ligand present in cell supernatants is a 13 kD protein,see FIG. 9., which migrates though gel with apparent molecular weight of13 kD. The protein with an apparent molecular weight of about 13 kDappears upon polyacrylamide gel electrophoresis as a smeared band whichmay indicate that the protein is glycosylated, enzyme modified, or thatthe band contains more than one protein species. Using mass spec andmaldi mass spec techniques, combined with homolgy to peptide sequencesin the Genbank database, it was determined that two fragments derivedfrom the 13 kD corresponded with a high degree of homology tobeta-lipotropin (Odell W, Wolfsen A, Bachelot I, and Hirose F, (1979)“Ectopic production of lipotropin by cancer” The American Journal ofMedicine 66; pgs. 631-638), which was previously known as beta-MSH(beta-melanotropin). Beta-lipotropin (beta-LPH: 98 amino acids or about10 kd) and ACTH (aa′ 130-169) are cleavage products ofproopiomelanocortin (Publisher Williams & Wilkins, chapter authors: FayeW, Lemke T, Williams D, Text book—Principles of Medicinal Chemistry;Fourth edition 1995) (POMC: 260 amino acids). Because these peptides areglycosylated, their apparent molecular weights can be altered from theiractual molecular weights. These cell surface receptor binding ligandscan be purified or produced using techniques known to those of skill inthe art. It is also possible that in certain situations an externalligand does not bind to and dimerize the MGFR portions of the MUC1receptor to trigger cell proliferation, but rather the receptors becomecovalently coupled to each other, for example by an enzyme thatcovalently links the two. One way this could be accomplished is by anenzyme that attaches an entity, such as a sugar group, to bothreceptors.

[0108] Because the portion of the MUC1 receptor that self-aggregates(IBR), and in doing so may protect intracellular signaling sequencesfrom participating in signaling cascades that induce proliferation, canbe cleaved and shed from the cell surface, it can be beneficial toidentify small molecules that interact with the MGFR portion of MUC1that remains cell-attached (MGFR). These small molecules that bind tothe portion of MUC 1 that remains cell-attached (MGFR) can then be usedin two ways. First, they can be used to block the remaining,cell-attached portion of the MUC1 receptor so that it cannot interactwith activating ligands that induce proliferation and metastasis. Forexample, a ligand to the cell-attached portion of MUC 1 may effectinductive multimerization of the receptor, causing a signaling cascadeinside the cell. Blocking binding of the ligand to the MGFR region caninhibit the signaling cascade that causes proliferation. Secondly, asdiscussed in more detail below, the small molecules can be polymerizedor attached to dendrimers to artificially cause preventative clusteringthe cell-attached MGFR portions of the MUC1 receptors and thus shieldthe cytoplasmic tails from interaction with intracellular signalingproteins.

[0109] The findings of the invention have important implications fordiagnostic and therapeutic procedures. For instance our finding that afragment of the MUC1 receptor that is close to the cell surfaceaggregates with itself, indicates that the position of enzyme cleavageis associated with receptor clustering, accessibility of adjacentportions of the receptor to putative ligands, and thus cancer. Agentsthat modulate the activity of this enzyme may be potent anti-canceragents. Additionally, an early diagnostic test for cancers thataberrantly express MUC1 may be based on detecting the portion of MUC1that self-aggregates (IBR) circulating in bodily fluids. This portion ofMUC1 includes part or all of the PSIBR (sequence shown in Table 1).Agents that bind to the portion (some or all of the PSMGFR sequence) ofMUC1 that remains attached to the cell surface after the release of theportion that self-aggregates (IBR—some or all of the PSIBR sequence) maybe potent anti-cancer drugs. In addition, agents that block binding ofthe natural ligand to the remaining portion after the release of theIBR, may also be useful as anticancer drugs. Drug candidates that targetportions of the MGFR, ie., sequences including some or all of the aminoacids contained within the PSMGFR, of the receptor can be used either asmonomers, to block the interaction of the MGFR portion of MUC1 withextracellular agents or biomolecules, or as a polymer, dendrimer, etc.to both block the interaction with external biomolecules and toartificially cluster the MGFRs. Another alternative agent, which can beused to artificially cluster the MGFRs is an IgM antibody raised againstthe MGFR or PSMGFR. This artifically-induced clustering may serve tokeep the cytoplasmic tails clustered to prevent interaction withintracellular signaling agents, thereby effecting preventativeclustering.

[0110] One aspect of the invention involves s novel drug screeningassays, that identify therapeutics that interfere with the proliferationof tumor cells that aberrantly express MUC1. The drug screen makes useof the new molecular target for cancer that is disclosed herein. Anotheraspect of the invention involves therapeutics identified by the drugscreen. Yet another aspect of the invention involves methods fordiagnosing MUC1⁺ cancers, which is based upon the mechanism elucidatedby the inventors.

[0111] One embodiment of the invention involves a drug screening assaywhich can rapidly identify agents that interrupt the interaction betweenthe MGFR and its ligand(s) and thus can be used as cancer therapeutics,(see Example 5a and FIG. 12 for details). The fact that an activatingligand(s) that binds to the MGFR portion of the MUC1 receptor can resultin inductive multimerization of the receptor, allows us to construct aconvenient drug-screening assay to identify compounds that inhibit thisinteraction or inhibit enzymes that modify the MGFR portion required forligand binding and thus inhibit the proliferation signal. In one assayof the invention, synthetic peptides which include much or all of theMGFR sequence are attached to nanoparticles such as gold colloids. Goldcolloids have the intrinsic optical property that when in a disperse,homogeneous solution, the solution appears pink, but when the colloidsare forced into close proximity, the solution turns blue. When celllysates or supernatants, which contain a ligand(s) that dimerizes thecolloid-attached peptides, are added, the colloids becomes aggregatedand the solution turns blue. Drug candidates that interrupt thisligand-receptor interaction are easily identified because they cause thesolution to remain pink.

[0112] As discussed above, it appears that the ligand that binds to theMGFR portion of the receptor to trigger inductive multimerization mayrecognize an enzyme-modified form of the receptor. Therefore theabove-described drug screening assay can identify compounds that: a)inhibit enzyme modification of the MGFR portion of the MUC1 receptor; b)bind to the MGFR region and block its interaction with an activatingligand of the MGFR portion; or c) bind to an activating ligand, such asa growth factor and block its interaction with the MGFR portion. Drugsthat act according to (a) or (b) are predicted to selectively inhibitMUC1-dependent cell proliferation, whereas drugs that act according to(c), on agents such as growth factors, are expected to inhibitproliferation of a variety of cell types, see Examples 5a-d.

[0113] Drugs that have been found by the above protocol to disrupt aninteraction between the MGFR portion of the MUC1 receptor and itsligand(s) are described in much greater detail below and include, forexample, calcimycin, fusaric acid, L-α-methyl-dopa, butylindazone,NS1619 or etomoxir. Additional drugs are described in greater detail inapplicant's co-pending U.S. Provisional Patent Application Serial Nos.60/317,302 and 60/317,314, both filed on Sep. 5, 2001 and entitledCompositions and Methods of Treatment of Cancer.

[0114] Agents so identified may be potent anti-cancer agents either inmonomeric form or as polymers or dendrimers. Drug libraries and peptidelibraries can be screened for molecules that inhibit binding of theligand to its target.

[0115] Alternatively, standard methods can also be used to identifyagents that interrupt the interaction between the MGFR and itsligand(s). These methods can be used to identify agents that bind: (1)to the MGFR portion of the MUC1 receptor, or (2) to one or more of itsligands. These methods include but are not limited to phage displaymethods, yeast two-hybrid system, sandwich assays, surface plasmonresonance-based assays, antibody-based assays, peptide bead assays fortesting with drug libraries, bead assays, GFP-reporter assays, and thelike. Ligands to the MGFR portion of the MUC1 receptor can be identifiedby a number of methods including using a peptide whose sequencecorresponds to some or all of the MGFR, ie. the PSMGFR peptide (SequenceID 7), as bait to fish out ligands. Another way to do this is to attacha signaling entity, such as GFP (green fluorescent protein), directly orindirectly to the ligand and attach the receptor-derived peptide to asolid support. Compounds that interfere with the interaction will causea loss of signal.

[0116] As an alternative to the natural ligand competition assaysdescribed above, direct binding assays can be employed to identify drugagents able to interact with the MGFR. Small molecules that bind to theremaining extracellular portion of cleaved MUC1 (MGFR region:exemplifiedby most or all of the PSMGFR peptide sequence), can be identified usingstandard methods (MALDI, western blotting, gel electrophoresis, ELISA,etc) or using colloid-colloid or colloid-bead binding assays. Forexample, in one embodiment, small molecules can be synthesized on beadsor attached to colloids either by attachment to a thiol and directincorporation into a SAM on the colloid or by EDC/NHS coupling ofmolecules containing a primary amine. A histidine-tagged peptideincluding the desired MUC1 (e.g. His-PSMGFR) sequence can be bound tocolloids and assayed for interaction with a second set of colloidspresenting a candidate drug. A color change from pink to blue wouldindicate an interaction between the MUC1 peptide and the small molecule.Alternatively, the MUC1-bearing colloids can be assayed for aninteraction with a small molecule attached to a bead. Red coloration ofthe bead surface would signify that the MUC1 peptide bound to thebead-immobilized small molecule.

[0117] Any drugs or small molecules identified as binding to the MUC1sequence can potentially be used to block binding of the remainingextracellular portion of cleaved MUC1 to its natural ligand, and canpotentially inhibit cancer growth.

[0118] In another embodiment, the above-described competition assays areemployed to identify “second generation” drug candidates by assayingsuch candidates for their ability to disrupt an interaction between theMGFR portion of the MUC1 receptor and a synthetic ligand found,according to the inventive methods described above, to bind to the MGFR,e.g. calcimycin, fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 oretomoxir. In this way, drugs that bind the MGFR with higher affinitythan the “first generation” drug can be identified. For example, inperforming the drug screen, a synthetic ligand such as calcimycin,fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 or etomoxir ismodified to facilitate attachment to surfaces, such as colloids. Apeptide derived from the MGFR region of the MUC1 receptor, such as theHis-PSMGFR, is attached to a second set of colloids. Direct bindingbetween the synthetic ligand and the MGFR peptide is confirmed, alibrary of drug candidates are then assayed for their ability to disruptthe interaction. It is not intended that this aspect of the invention belimited to the competition techniques or assays explicitly describedherein. Several techniques, including standard methods, couldpotentially be used to detect binding between the MGFR and a syntheticligand or drug, then competitive inhibition of binding by a drugcandidate.

[0119] Another aspect of the invention is a drug screening assay foridentification of drugs that can be useful for prevention and/ortreatment of cancer by altering the cleavage state of MUC 1 receptors oncells. In such assays, described in more detail below, cultured cellsare exposed to candidate drugs. The cleavage state of MUC 1 in the cellsis determined, optionally as a function of time and/or dosage or otherconditions involving exposure to the drugs. These cells can be derivedfrom a particular patient, or can be tumor-associated ornon-tumor-associated cell lines. Customized therapeutic protocols can bedetermined for a particular patient in this manner. The inventioninvolves, in one aspect, treating a patient with a drug, as discused ingreater detail below, shown to affect the cleavage state of MUC 1 of thepatient's cells in a manner that prevents, inhibits, or reverses cancer.

[0120] Because it is suspected that the incorrect cleavage of MUC1 onthe surface of the cell causes the cascade leading to proliferation andtumorigenesis, it would be advantageous to test candidate drugs in awhole cell assay for their ability to affect enzyme cleavage or theposition of enzyme cleavage of MUC1. Drugs can be screened for theirability to effect MUC1 cleavage, either directly or indirectly. This canenable the identification of upstream effectors of MUC1 cleavage. Cellsor tissue samples can be assayed for MUC1 cleavage potential in severalways. For example, cells or a tissue sample can be treated with a drugcandidate and grown for some period of time. Colloids bearing anantibody, natural ligand, or small molecule that binds to either thecleaved portion of MUC1, or the remaining extracellular portion (plus orminus a signaling moiety) can be added and allowed to bind to the cellsor tissue sample. The expression of cleaved MUC1 or uncleaved MUC1 onthe cell surface as compared to a control sample, not treated with thecandidate drug, would indicate whether the drug candidate effected MUC1expression or cleavage.

[0121] Alternatively, MUC1 expressing cells can be assayed for MUC1cleavage in the presence of a drug candidate by testing the surroundingcell growth media for the presence of cleaved MUC1 or the potential ofthe cleaved portion to self-aggregate. For example, cells expressingMUC1 would be treated with a drug candidate suspected of interferingwith enzyme cleavage. After some incubation period, the cell media wouldbe removed and tested for its aggregation potential, i.e. to determinewhether the self-aggregating portion of MUC1 was contained within theshed fragment. The aggregation potential of peptides released into thecell media is tested by adding colloids bearing an antibody to asequence distal from the self-aggregating portion, but not a repeatsequence. In this way, antibody-presenting colloids would attach toupstream regions of MUC1. If the self-aggregating region was alsoattached to the released fragment, then this would cause aggregation ofthe attached colloids and a solution change from pink to blue wouldresult. Accordingly, one aspect of the invention provides a composition(drug or agent) that, in contrast to preventing inductivemultimerization of cleaved or modified MUC1 receptors as discussedpreviously, instead prevents disease-associated MUC 1 cleavage ormodification itself. MUC 1 cleavage is an enzymatic process. Thedetermination of the mechanism involved by the inventors can lead toidentification of a drug that inhibits disease-associated cleavage.

[0122] As mentioned above, another aspect of the invention provides anagent that binds together MGFR portions of MUC 1 followingdisease-associated cleavage to effect preventative clustering of thereceptors. The agent can be any species that includes multiple siteseach able to bind to a MFGR portion, and immobilized with respect toeach other. E.g. a polymer or dendrimer or other continuous entity caninclude multiple sites each able to bind to a MGFR portion, causingclustering of these portions or other structural constraint thatinhibits their association with factors that promote cell proliferation.Alternatively, IgM-type monoclonal or polyclonal antibodies raisedagainst the MGFR or PSMGFR could be utilizied. Each anti-MGFR IgMantibody could be able to aggregate ten MGFRs on the cell surface toform preventative clusters.

[0123] In addition, some or all of the above-identified ligand speciesthat bind to the MGFR can be modified to allow the ligands to act as atargeted delivery agent by attaching a cytotoxic drug or other agent(e.g. a radioactive substance) able to selectively kill cells to whichthe ligands become immobilized. In addition, synthetic ligands, such ascalcimycin, fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 oretomoxir, discussed in more detail below, that were found to bind to theMGFR portion of the MUC1 receptor can similarly be modified with othertherapeutic agents. In this way, such a therapeutic can be directed tothe tumor cells. For example, an agent that binds to the MGFR region ofthe MUC1 receptor can be modified with a radioactive substance todestroy tumor cells that aberrantly express the MUC1 receptor. Othertoxic substances, such as ricin, as well as other therapeutics, can beattached to agents that bind the MGFR. Alternatively, identified ligandspecies that bind to the MGFR could be modified to present a imagingagent for use in diagnostic imaging of MUC 1⁺ tumors and metastases.Such ligands can also, alternatively, be modified to act as drugs thatcan be useful for prevention and/or treatment of cancer. In oneembodiment, a ligand, which in its unmodified form binds to multipleMGFRs causing inductive multimerization, is modified to remove orde-activate all but one of its active binding sites for MGFR, such thateach modified ligand is able to bind to only a single receptor. Inanother embodiment, individual ligand molecules/peptides are modifiedsuch that they are immobilized with respect to additional ligandmolecules/peptides also able to bind MGFR, e.g. through covalentcoupling, non-covalent coupling, co-immobilization with respect to asubstrate, etc., such that the modified, multi-unit ligand is able theffect preventative clustering of the receptors to which it binds.

[0124] Identification of the ligand(s) for the portion of MUC1 thatremains bound to the cell after cleavage can allow, as discussed furtherbelow, for development of powerful assays to screen for drugs thatdisrupt this interaction. Interaction of potential binding partners withthe extracellular portion of MUC1 that remains after cleavage can bestudied both by conventional techniques (western blotting, ELISA, MALDI,etc.) and using our colloid-colloid color change assay or colloid-beadcoloration assay. The peptide sequence of the remaining extracellularportion of MUC1 can be attached to beads or colloids via a histidinetag. Potential binding partners can be histidine-tagged and attached toa second set of colloids (or beads) and assayed for binding to thecolloid-immobilized portion of MUC1. Alternatively, potential bindingpartners can be attached to beads or colloids by EDC/NHS coupling or canbe nonspecifically adsorbed to beads for the assay. An interactionbetween the MUC1 peptide and the potential binding partner can bedetected by either a change in solution color (for the colloid-colloidassay) or by agglomeration of the colloids onto the bead, causing thebead to appear red (for the colloid-bead assay). An entire cDNA librarycan be screened using this technique in a short period of time toidentify the natural ligand of the remaining extracellular MUC1. (seePCT/US00/01997, WO 00/34783, 09/631,818, and “Detection of BindingSpecies with Colloidal and Non-Colloidal Structures”, filed Nov. 15,2000, incorporated above).

[0125] The discoveries presented herein: (1) that the IBR of MUC1self-aggregates; (2) that an antibody that dimerizes adjacent MGFRportions of the MUC1 receptor leads to proliferation of MUC1 presentingtumor cells; and (3) that proliferation of MUC1 presenting tumor cellscan be inhibited by treatment with agents that target the MGFR and blockthe MGFR against interaction with a ligand, are consistent with amechanism in which, in a healthy cell, MUC1 cleavage occurs such thatenough of the IBR remains on the cell that MUC1 remains clustered, andthe MGFR is inaccessible to ligands such as growth factors, and in atumor cell, MUC1 cleavage occurs such that enough of the IBR is cleavedfrom the cell such that MUC1 does not remain clustered, and the MGFR isaccessible to ligand interaction. This leads to diagnostics, provided bythe present invention, in which the shed portion of MUC1 is analyzed todetermine the degree of IBR present.

[0126] The above-mentioned mechanistic model predicts that in a subjectwith a MUC1-dependent tumor or who is prone to developing such a tumor,the portion of the MUC1 receptor that is shed will contain the IBRregion of the receptor, leaving the MGFR portion of the receptoraccessible for interactions with ligands and growth factors. An earlydiagnostic would consist of detecting the IBR region in the portion ofthe MUC1 receptor which is shed.

[0127] In one embodiment, loss of aggregation of MUC1 receptors canresult from the “cleavage state” of MUC1. “Cleavage state” defines theresult of cleavage of MUC 1. The cleavage state will differ between ahealthy cell and a cell with tumor potential. The cleavage statedetermination can involve determining whether cleavage occurs in amanner such that the normal interaction between the IBRs of neighboringreceptors is disrupted (these regions no longer remain bound to eachother at the cell) and MUC 1 is free to spread across the cell surface.More specifically, determination of the cleavage state can includedetermining a site of cleavage of MUC 1, determining the identity of aportion of MUC 1 that remains at a cell following cleavage, determiningthe identity of a portion of MUC 1 that is separated from the cellfollowing cleavage (“cleaved or shed portion”), determining theaccessibility of MGFR, or a combination.

[0128] In one embodiment, an assay is provided that can determinewhether the MUC1 IBR remains fastened to the cell, or is separated fromthe cell upon cleavage. The product of MUC1 cleavage from the cell isexposed to at least one surface adapted to bind the IBR and to anothersurface and/or a signaling entity. Generally, an assay as described inWO 00/43791 or WO 00/34783 can be used. In a specific example,antibodies to a portion of MUC1 that would remain fastened to the IBR ifthe IBR is cleaved from the cell, such as antibodies to the repeatsdomain, are fastened to colloids. Exposure of these colloids to the MUC1cleavage products will allow the IBR regions to self-aggregate which inturn will result in colloid/colloid aggregation (color change to blue)for cases in which the IBR portion is separated from the cell. This isbecause each colloid will fasten to a MUC 1 region which is connected toan IBR which is fastened to another IBR which is in turn fastened toanother colloid.

[0129] The discovery that tumor cells can be treated with an agent thatbinds to the MGFR of MUC1, or a ligand of MGFR, in a manner thatinhibits cell proliferation leads to the conclusion that, in a diseasedcell (a cancerous cell or a cell with potential for becoming cancerous),cleavage of MUC 1 occurs in a manner that allows MGFR to interact withat least one ligand in a manner that promotes tumorigenesis or cancer.Interaction with the ligand may be due to cleavage that disrupts bindingbetween different MUC 1 molecules at the cell surface, either viaseparation from the cell of the IBR during cleavage, or cleavage withinthe IBR at a location that frees MUC 1 molecules from each other.

[0130] In one aspect of the invention, a diagnostic is provided whereinan amount of cleavage of a cell surface receptor IBR from the cellsurface is determined. This involves the determination of the amount ofcell surface receptor IBR that is separated from the cell surface uponcleavage, which can be relative to the level determined in past samples,the level in control samples, or can be determined as a ratio of theamount of IBR to the amount of a constant region of the receptor in asample. The constant region is any non-repeating sequence which isN-terminal to the boundary of the PSIBR, that is, this region is presentin a 1:1 ratio with the PSIBR prior to cleavage—determination of theratio of IBR to constant region subsequent to cleavage indicates theextent to which the IBR is cleaved and separated from the cell. Theamounts of various receptor regions may be determined with any type ofbinding assay, e.g. an antibody-binding assay. For example, antibodiesthat specifically bind to the constant region or the repeats may beattached to surfaces (e.g. magnetic beads) to preconcentrate shed MUC 1receptors prior to determining levels of IBR present. Then, for example,after pre-concentration of circulating MUC 1 receptors, antibodies tothe IBR and antibodies to the constant region can be allowed to bind tothe cleaved receptors, and determination of the ratio of binding ofthese antibodies reveals the ratio of IBR present relative to constantregion present in the cleaved receptors, which in turn reveals theamount of cleavage that occured in a manner that caused separation ofthe IBR from the cell. A ratio that displays a trend toward less thanone (less than a 1:1 ratio of IBR relative to constant region present)for detecting IBR at a cell surface is an indicator of the presence of atumor or the potential for the development of a tumor. A ratio thatapproaches 1:1 when detecting these regions in shed receptors islikewise an indicator of cancer potential. This determination canindicate potential for tumor formation, existence of a tumor,progression of tumorigenesis, etc., and can thereby serve as adiagnostic and/or a evaluator of treatment for tumorigenesis

[0131] Another diagnostic aspect of the invention involves determininglevels of shed IBRs in sample from a subject. Methods for suchdetermination can include determining the aggregation potential of thecirculating shed receptors, for example via colloid-binding assays suchas a colloid-colloid assay or a colloid bead assay (See above discussionand Examples, below). Alternative techniques involve determining thepresence of the IBR using antibody probing assays, hybridization, PCRReverse Transcriptase PCR (rtPCR), Ligase Chain Reaction (LCR), cyclingprobe technology, etc. In a preferred embodiment of the invention, thecell surface receptor is MUC1.

[0132] The determination, in a blood sample, of the amount of cleavedreceptor carrying IBR, either involving antibody binding ratios, colloidbinding assays, or the like can be made on a bodily fluid sample, suchas a blood sample and optionally compared with other samples (e.g. tomonitor the subject's progression of tumorigenesis or progression fortreatment of the same) and/or controls.

[0133] Alternatively, biopsy specemins can be studied, or tissue can bestudied interoperatively (e.g. tissue at a surgical site can be studiedwithout removal of the tissue from the subject). In either of thesestudies, a primary indicator of tumorigenesis or potential fortumorigenesis is the amount of MGFR at a cell surface accessible tointeraction with external agents such as growth factors, etc. Thisdetermination can be made, for example, by determining the amount of anantibody to the MGFR region that binds to the sample, either usingstandard antibody binding study techniques, or by exposing the sample tocolloids to which antibodies specific to the MGFR region have beenimmobilized and determining binding of the colloids to the samples usingtechniques described in International patent publication numbers WO00/34783 and WO 00/43791, referenced above. In another technique(perhaps more suited for an excised sample), antibodies to the MGFRregion and to the IBR can be exposed to the sample and a determinationmade of the ratio of binding of each to the sample. A healthy samplewill exhibit little or no antibody binding to the MGFR region. A sampleindicating tumerigenesis or potential for tumorigenesis will show anon-zero ratio of MGFR antibody binding to IBR antibody binding.

[0134] Whether shed MUC1 contains the IBR can be identified by methodsknown to those of ordinary skill in the art, including ELISA andcolloidal assays as described in international patent application serialno. PCT/US00/01997, filed Jan. 25, 2000, entitled “Rapid and SensitiveDetection of Aberrant Protein Aggregation in NeurodegenerativeDiseases”, published as no. WO 00/43791, and U.S. patent applicationSer. No. 09/631,818, filed Aug. 3, 2000, entitled “Rapid and SensitiveDetection of Protein Aggregation”, by Bamdad et al. In a preferredembodiment of the invention, the cell surface receptor is MUC1.

[0135] Another aspect of the invention involves determining the site ofcleavage of the MUC1 receptor from a cell surface (rather than theamount of IBR in a shed portion) in a sample from a subject to evaluatecancer, or the potential to develop cancer in a subject. Determinationof the site of cleavage will give information as to whether the IBRremains on the cell surface, or was shed from the cell surface, givingindication of cancer or tumorigenesis or the potential for either, asdiscussed above. Determining the site of cleavage can be accomplished byusing enzyme-amplification methods such as PCR. Specifically, usingalternative primer sites in PCR amplification of shed MUC1 fromsubject's sample will indicate where cleavage occurred.

[0136] In one aspect of the invention, differences in pre- andpost-treatment levels of cleaved cell surface receptor IBR, or cellsurface receptor IBR at the surface of a cell, in cancer cells ortissues may be used to diagnose cancer in a subject or assess theeffectiveness of treatment in a cancer patient. In a preferredembodiment the cell surface receptor is MUC1.

[0137] Comparison of the levels of the above-mentioned regions withlevels from subjects known to be free of cancer may allow determinationof the presence of cancer in the subject. An example, although notintended to be limiting, is that a determination of the presence ofelevated levels of cleaved cell surface IBR in a sample from a subject,when compared to a level determined in samples from control subjects,may suggest the presence of cancer in the subject with elevated levels.Such methods of comparing levels of cancer-associated markers between asample from a subject and a control sample for diagnostic purposes wouldbe understood by one of ordinary skill in the medical arts. Examples ofsuch methods include Western blotting, ELISA, antibody precipitation,PCR, LCR, rtPCR, cycling probe technology, and colloidal assays asdescribed in international patent application serial no. PCT/US00/01997,filed Jan. 25, 2000, entitled “Rapid and Sensitive Detection of AberrantProtein Aggregation in Neurodegenerative Diseases”, published as no. WO00/43791, and U.S. patent application Ser. No. 09/631,818, filed Aug. 3,2000, entitled “Rapid and Sensitive Detection of Protein Aggregation”,by Bamdad et al. In a preferred embodiment the cell surface receptor isMUC1.

[0138] In another aspect of the invention, the cleavage state of MUC 1can be used to determine progression or regression of a subject's cancerover time. The cleavage state also can be used to assess treatmentparameters including, but not limited to: dosage, method ofadministration, timing of administration, and combination with othertreatments as described herein.

[0139] Another aspect of the invention involves extremely early-stagecancer diagnosis. This aspect involves identification of patients whomay be at risk for developing tumor or cancer associated with abnormalcleaveage of MUC1. These patients may not have developed tumors, but mayexhibit a cleavage state indicative of a condition that can lead tocancer. In some instances, the subjects will already be undergoingtreatment for cancer, while in other instances the subjects will bewithout present cancer treatment. A test for a genetic predisposition tocancers characterized by aberrant MUC1 expression of the invention isbased on detecting genetic alterations in the MUC1 cleavage enzyme(s),over expression of MUC1 activating ligands, and/or overexpression ofenzymes that modify the MGFR portion of the receptor.

[0140] The fact that elevated levels of cleaved MUC1 are found in theblood of cancer patients is the basis for a blood test for breastcancer, which is not described herein. MUC1 is cleaved by at least oneenzyme and may be cleaved at more than one site before it is releasedinto the blood stream. Several protease cleavage sites within the MUC1sequence are predicted. Predicted sites for enzyme cleavage are at ornear amino acid 540, 528, 530, 542, and 550 (numbers are as listed inAndrew Spicer et al., J. Biol. Chem Vol 266 No. 23, 1991 pgs.15099-15109; these amino acid numbers correspond to numbers 1100, 1088,1090, 1102, 1110 of Genbank accession number P15941; PID G547937; BoshelM., et al, BBRC vol. 185 pgs 1-8; Hilkens J., et al, Journal ofBiological Chemistry vol 267 pgs 6171-6177). The exact site of cleavagemay vary depending on cell type or in response to a disease. The enzymethat cleaves MUC1 may be a membrane-associated enzyme, since theclustered IBRs of the MUC1 receptor limit access to the cleavage sites.

[0141] One aspect of the invention is the identification of compoundsthat directly bind to the PSMGFR portion of the receptor. Therefore, asensitive method for diagnosing early tumors is to administer to thepatient, compounds that bind to the PSMGFR region that have also beenderivatized with contrast or imaging agents. These compounds willagglomerate onto tumors wherein this portion of the MUC1 receptor isaccessible. Compounds described herein that bind to the PSMGFR region aswell as other compounds that can be identified using methods of theinvention can be readily modified to carry imaging agents. Such imagingagents may include but are not limited to, technetium, rhenium, ¹²³I,and other contrast agents or radioactive entities commonly used inimaging techniques. Imaging techniques include but are not limited tosingle photon computed tomography (SPECT), MRI, microscopy and the like.In some applications, an attached colloid can act as an imaging agent.Since the carrier for the imaging agent can also be a therapeutic, thistechnique can combine an early diagnostic with a directed therapeutic.

[0142] As referred to previosly, one aspect of the invention is directedto methods for treating a subject diagnosed with or at risk ofdeveloping a cancer or tumor characterized by the aberrant expression ofMUC1. The treatments of the present invention involve the use of drugsor “agents” as described herein. That is, one aspect involves a seriesof compositions useful for treatment of cancer or tumor characterized bythe aberrant expression of MUC1, including these compositions packagedin kits including instructions for use of the composition for thetreatment of such conditions. That is, the kit can include a descriptionof use of the composition for participation in any biological orchemical mechanism disclosed herein that is associated with cancer ortumor. The kit also can include instructions for use of a combination oftwo or more compositions of some embodiments of the invention.Instructions also may be provided for administering the drug orally,intravenously, or via another known route of drug delivery. These andother embodiments of the invention can also involve promotion of thetreatment of cancer or tumor according to any of the techniques andcompositions and combinations of compositions described herein.

[0143] In one set of embodiments, patients can be treated withcompositions of the invention even though the patients exhibitindication for treatment of one of the compositions of the invention fora condition different from cancer or tumor, including conditions thatcan be unrelated to cell proliferation or conditions that can accompanycell proliferation, cancer, or tumor. That is, if a composition of theinvention is known for treatment of a different condition, someembodiments of the present invention also involve use of thatcomposition for treatments that accompany cell proliferation, cancer, ortumor disease where indicated. These and other embodiments of theinvention can include such treatment where the dosage, deliverytechnique or vehicle, combination with other pharmaceutical compositionsor lack of combination with other pharmaceutical compositions, rate ofadministration, timing of administration, or other factor differs fromthe use of the composition for treatment of the condition different fromcell proliferation, cancer, or tumor. In another set of embodiments,treatment of cell proliferation, cancer, or tumor with compositions ofthe invention may occur under conditions that are similar to or overlapthe use of compositions of the invention for treatment of a differentcondition, but the compositions of the invention are promoted fortreatments that accompany cell proliferation, cancer, or tumor orincludes instructions for treatments that accompany cell proliferation,cancer, or tumor as mentioned above. As used herein, “promoted” includesall methods of doing business including methods of education, hospitaland other clinical instruction, pharmaceutical industry activityincluding pharmaceutical sales, and any advertising or other promotionalactivity including written, oral, and electronic communication of anyform, associated with compositions of the invention in connection withtreatments that accompany cell proliferation, cancer, or tumor.“Instructions” can and often do define a component of promotion, andtypically involve written instructions on or associated with packagingof compositions of the invention. Instructions also can include any oralor electronic instructions provided in any manner. The “kit” typically,and preferably, defines a package including both any one or acombination of the compositions of the invention and the instructions,but can also include the composition of the invention and instructionsof any form that are provided in connection with the composition in amanner such that a clinical professional will clearly recognize that theinstructions are to be associated with the specific composition.

[0144] Subjects for whom certain treatment methods of the invention(with specific compositions directed toward cell proliferation, cancer,or tumor) are not intended are those who are diagnosed with a conditionwhich may already call for treatment with the specific composition.Accordingly, one aspect of the invention involves treatment of cellproliferation, cancer, or tumor with a specific composition disclosedherein for that purpose, not in combination with another agent where theother agent has been taught previously for use in treatment of cellproliferation, cancer, or tumor itself. Another embodiment involvestreatment of cell proliferation, cancer, or tumor with this specificcomposition alone, not in combination with any other active agent.Another embodiment involves treatment of cell proliferation, cancer, ortumor with this specific composition where the use of the composition inthe treatment is specifically instructed (through, e.g. writteninstructions that can accompany the composition) for the treatment ofcell proliferation, cancer, or tumor. In a preferred embodiment of thisaspect, the invention involves treatment of cell proliferation, cancer,or tumor with the specific composition where the use of the compositionin the treatment is specifically instructed to affect a mechanismassociated with cell proliferation, cancer, or tumor as disclosedherein.

[0145] In yet another set of embodiments, the drugs and agents of theinvention can be used for the purpose of disease prevention. In thiscontext, the invention is particularly directed to a patient populationnever before treated with drugs useful according to certain methods ofthe invention, including patients who are not suffering from cellproliferation, cancer, or tumor and who may or may not be presentlyindicating susceptibility to cell proliferation, cancer, or tumor. Inother words, the preventative treatment preferably is directed topatient populations that otherwise are free of disease symptoms thatcall for active treatment with any of the drugs described herein asuseful according to the invention.

[0146] In one aspect, the invention involves the discovery thatcalcimycin, fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 andetomoxir interrupt the interaction of MGFR with its ligand(s) thatotherwise would bind to MGFR and promote tumorigensis. In this aspect,the invention involves treatment of subjects associated with tumor orcancer associated with aberrant expression of MUC1 with these agents ora combination. These compounds were identified when a drug library wasscreened using the in vitro color change colloid aggregation assaydescribed in Example 5a. These compounds were then tested in a wholecell assay to determine if they produced the desired activity, namely ifthey inhibited cell proliferation by interfering with the MGFR-ligandinteraction. All of the compounds inhibited cell proliferation, butroughly half of the compounds were toxic to both tumor cells thatpresented the MUC1 receptor as well as cells that did not present thisreceptor. As discussed herein, the drug screen described in Example 5adoes not differentiate among drugs that inhibit cell proliferation by:(a) binding to or otherwise blocking the activity of a MUC1-associatedactivating ligand(s), such as growth factors; b) directly binding to theMGFR portion of the MUC1 receptor and blocking its interaction with itsactivating ligands; or (c) inhibiting the activity of enzymes thatmodify the MGFR portion of the Muc1 receptor. Drugs that act accordingto the mode of action described in (a) will not be selective forMUC1-presenting cells and are likely to be somewhat cytotoxic since theyinhibit essential growth factors. Drugs that act according to (b) and(c) will selectively inhibit the proliferation of MUC1-presenting cellsand further, those that directly bind to the MGFR portion will havelittle or no toxic effects. Fusaric acid, L-αmethyl-dopa and etomoxirselectively inhibited proliferation of tumor cells presenting MUC1 whileleaving control cells unaffected, see FIG. 13.

[0147] In one embodiment, the subject to be treated with the aboveagents can be otherwise free of signs, symptoms or evidence of disordersfor which the agents of the invention would normally be or havepreviously been described. Preferably, the subject is otherwise free ofsymptoms calling for treatment involving the use of at least any one ofcalcimycin, fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 andetomoxir, alone or in combination with each other or with otherpharmaceutically acceptable substances. For example, calcimycin, fusaricacid, L-α-methyl-dopa, butylindazone, NS1619 and etomoxir may have beensuggested for treatment of subjects having certain diseases; thus, inone embodiment, the preferred subjects are free of those disease forwhich the agents of the present invention have been previouslyprescribed.

[0148] Fusaric Acid.

[0149] Subjects for whom the methods of the invention involvingtreatment with fusaric acid are not intended are those diagnosed withdiseases which already call for treatment with fusaric acid, but wherethe call for treatment with fusaric acid did not specifically call fortreatment directed toward tumors or cancers associated with theabherrant expression of MUC1, particularly in the dosages or otherspecific protocols described previously in U.S. Pat. No. 6,127,393.Specific diseases listed in U.S. Pat. No. 6,127,393 include skin cancer,breast cancer, prostate cancer, cervical cancer, colon cancer, livercancer and lung cancer. In one embodiment, the methods of the presentinvention involve treatment with fusaric acid in dosages lower than thatdescribed in U.S. Pat. No. 6,127,393, as evidenced by graphs of FIG. 13which are analogous to FIGS. 3A-3C in U.S. Pat. No. 6,127,393 whichdepict an amount of fusaric acid needed to inhibit cell growth.

[0150] In one embodiment, the invention provides a fusaric acidtreatment in a lower dosage to provide a less than daily administrationregimen. For example, the fusaric acid can be provided every other dayor once weekly.

[0151] Etomoxir.

[0152] Subjects for whom the methods of the invention involvingtreatment with etomxir are not intended are those diagnosed withdiseases which already call for treatment with etomoxir, particularlythose subjects who have diseases associated with chronic heart failurecalling for treatment with etomoxir. Such diseases include failingcardiac hypertrophy associated with an inadequate sarcoplasmic reticulumfunction.

[0153] NS1619.

[0154] Although NS1619 is currently known as a biochemical tool as aK(ca) channel activator, subjects for whom the methods of the inventioninvolving treatment with NS1619 are not intended are those diagnosedwith diseases which already call for treatment with NS1619 requiringK(ca) channel modulation.

[0155] Calcimycin.

[0156] Calcimycin is an ionophorous, polyether antibiotic fromstreptomyces chartreusensis. Calcimycin binds and transports cationsacross membranes and uncouples oxidative phosphorylation whileinhibiting atpase of rat liver mitochondria. The substance is usedmostly as a biochemical tool to study the role of divalent cations invarious biological systems. Subjects for whom the methods of theinvention involving treatment with calcimycin are not intended are thosediagnosed with diseases which already call for treatment with calcimycinin this function.

[0157] Butylindazone.

[0158] R(+)-butylindazone is a KCl cotransport inhibitor, and subjectsfor whom the methods of the invention involving treatment withbutylindazone, specifically R(+)-butylindazone, are not intended arethose diagnosed with diseases which already call for treatment withbutylindazone requiring inhibition of KCl cotransport.

[0159] The method comprises administering to the subject calcimycin,fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 and etomoxir in anamount effective to provide a medically desirable result. In oneembodiment, the method comprises administering to the subject any one ofcalcimycin, fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 andetomoxir in an amount effective to lower the risk/prevent/reduce/inhibittumors or cancer associated with aberrant expression of MUC1.

[0160] The effective amount will vary with the particular conditionbeing treated, the age and physical condition of the subject beingtreated, the severity of the condition, the duration of the treatment,the nature of the concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. For example, in connection with tumor or cancerassociated with abherrant expression of MUC1, an effective amount isthat amount which prevents interaction of MGFR with its ligand thatotherwise would promote cell proliferation (for agents that actaccording to that mechanism, including calcimycin, fusaric acid,L-α-methyl-dopa, butylindazone, NS1619 and etomoxir).

[0161] According to alternate mechanisms of drug activity, an effectiveamount is that amount which maintains self-aggregation of MUC1 receptors(for agents such as polymers or dendrimers that act according to thatmechanism). Alternatively, an effective amount is one which reduceslevels of cleaved MUC1 IBRs, or maintains low levels of cleaved MUC1 BRs(for agents that act according to that mechanism). Likewise, aneffective amount for treatment would be an amount sufficient to lessenor inhibit altogether the levels of cleaved MUC1 IBR (for agents thatact according to that mechanism) so as to slow or halt the developmentof or the progression of tumor or cancer associated with aberrantexpression of MUC1. It is preferred generally that a maximum dose beused, that is, the highest safe dose according to sound medical judgment

[0162] When used therapeutically, the agents of the invention areadministered in therapeutically effective amounts. In general, atherapeutically effective amount means that amount necessary to delaythe onset of, inhibit the progression of, or halt altogether theparticular condition being treated. Generally, a therapeuticallyeffective amount will vary with the subject's age, condition, and sex,as well as the nature and extent of the disease in the subject, all ofwhich can be determined by one of ordinary skill in the art. The dosagemay be adjusted by the individual physician or veterinarian,particularly in the event of any complication. A therapeuticallyeffective amount typically varies from 0.01 mg/kg to about 1000 mg/kg.It is expected that does ranging from 1-500 mg/kg, and preferably dosesranging from 1-50 mg/kg will be suitable. In other embodiments, theagents will be administered in doses ranging from 1 μg/kg/day to 10mg/kg/day, with even more preferred doses ranging from 1-200 μg/kg/day,1-100 μg/kg/day, 1-50 μg/kg/day or from 1-25 μg/kg/day. In otherembodiments, dosages may range from about 0.1 mg/kg to about 200 mg/kg,and most preferably from about 0.2 mg/kg to about 20 mg/kg. Thesedosages can be applied in one or more dose administrations daily, forone or more days.

[0163] The agent of the invention should be administered for a length oftime sufficient to provide either or both therapeutic and prophylacticbenefit to the subject. Generally, the agent is administered for atleast one day. In some instances, the agent may be administered for theremainder of the subject's life. The rate at which the agent isadministered may vary depending upon the needs of the subject and themode of administration. For example, it may be necessary in someinstances to administer higher and more frequent doses of the agent to asubject for example during or immediately following a event associatedwith tumor or cancer, provided still that such doses achieve themedically desirable result. On the other hand, it may be desirable toadminister lower doses in order to maintain the medically desirableresult once it is achieved. In still other embodiments, the same dose ofagent may be administered throughout the treatment period which asdescribed herein may extend throughout the lifetime of the subject. Thefrequency of administration may vary depending upon the characteristicsof the subject. The agent may be administered daily, every 2 days, every3 days, every 4 days, every 5 days, every week, every 10 days, every 2weeks, every month, or more, or any time there between as if such timewas explicitly recited herein.

[0164] In one embodiment, daily doses of active compounds will be fromabout 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It isexpected that oral doses in the range of 50 to 500 milligrams/kg, in oneor several administrations per day, will yield the desired results.Dosage may be adjusted appropriately to achieve desired drug levels,local or systemic, depending upon the mode of administration. In theevent that the response in a subject is insufficient at such doses, evenhigher doses (or effective higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

[0165] Preferably, such agents are used in a dose, formulation andadministration schedule which favor the activity of the agent and do notimpact significantly, if at all, on normal cellular functions.

[0166] As noted, different drugs act according to different mechanisms.Drugs according to one mechanism interfere with MGFR binding to atumorigenesis-promoting ligand, and do so to a particular degreerelative to natural conditions for the subject in the absence of thedrug. Drugs according to another mechanism reduce overall cleavage ofMUC1, and do so to a particular degree relative to natural conditionsfor the subject in the absence of the drug. Drugs according to anothermechanism maintain self-aggregation of MUC1 receptors, and do so to aparticular degree relative to natural conditions for the subject in theabsence of the drug. In one embodiment, the degree of activity of thedrug is at least 10%. In other embodiments, the degree of activity ofthe drug is as least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0167] When administered to subjects for therapeutic purposes, theformulations of the invention are applied in pharmaceutically acceptableamounts and in pharmaceutically acceptable compositions. Such apharmaceutical composition may include the agents of the invention incombination with any standard physiologically and/or pharmaceuticallyacceptable carriers which are known in the art. The compositions shouldbe sterile and contain a therapeutically effective amount of the agentin a unit of weight or volume suitable for administration to a patient.The term “pharmaceutically-acceptable carrier” as used herein means oneor more compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration into a human or otheranimal. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the molecules ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficacy. Pharmaceutically acceptable further means anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

[0168] Such preparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticingredients. When used in medicine the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic,tartaric, citric, methane sulfonic, formic, malonic, succinic,naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

[0169] Suitable buffering agents include: acetic acid and a salt (1-2%W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5%W/V); and phosphoric acid and a salt (0.8-2% W/V).

[0170] Suitable preservatives include benzalkonium chloride (0.003-0.03%W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) andthimerosal (0.004-0.02% W/V).

[0171] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular combination ofdrugs selected, the severity of the cancer condition being treated, thecondition of the patient, and the dosage required for therapeuticefficacy. The methods of this invention, generally speaking, may bepracticed using any mode of administration that is medically acceptable,meaning any mode that produces effective levels of the active compoundswithout causing clinically unacceptable adverse effects. Such modes ofadministration include oral, rectal, topical, nasal, other mucosalforms, direct injection, transdermal, sublingual or other routes.“Parenteral” routes include subcutaneous, intravenous, intramuscular, orinfusion. Direct injection may be preferred for local delivery to thesite of the cancer. Oral administration may be preferred forprophylactic treatment e.g., in a subject at risk of developing acancer, because of the convenience to the patient as well as the dosingschedule.

[0172] Chemical/physical vectors may be used to deliver the agents ofthe invention to a target (e.g. cell) and facilitate uptake thereby. Asused herein, a “chemical/physical vector” refers to a natural orsynthetic molecule, other than those derived from bacteriological orviral sources, capable of delivering the agent of the invention to atarget (e.g. cell).

[0173] A preferred chemical/physical vector of the invention is acolloidal dispersion system. Colloidal dispersion systems includelipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. A preferred colloidal system of the inventionis a liposome. Liposomes are artificial membrane vessels which areuseful as a delivery vector in vivo or in vitro. It has been shown thatlarge unilamellar vessels (LUV), which range in size from 0.2-4.0.mu.can encapsulate large macromolecules. RNA, DNA, and intact virions canbe encapsulated within the aqueous interior and be delivered to cells ina biologically active form (Fraley, et al., Trends Biochem. Sci., v. 6,p. 77 (1981)). In order for a liposome to be an efficient gene transfervector, one or more of the following characteristics should be present:(1) encapsulation of the gene of interest at high efficiency withretention of biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation.

[0174] Liposomes may be targeted to a particular (e.g. tissue), such as(e.g. the vascular cell wall), by coupling the liposome to a specificligand such as a monoclonal antibody, sugar, glycolipid, or protein.

[0175] Liposomes are commercially available from Gibco BRL, for example,as LIPOFECTIN™. and LIPOFECTACE™., which are formed of cationic lipidssuch as N-[1-(2,3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, V. 3, p. 235-241 (1985).

[0176] In one particular embodiment, the preferred vehicle is abiocompatible micro particle or implant that is suitable forimplantation into the mammalian recipient. Exemplary bioerodibleimplants that are useful in accordance with this method are described inPCT International application no. PCT/US/03307 (Publication No. WO95/24929, entitled “Polymeric Gene Delivery System”, claiming priorityto U.S. patent application Ser. No. 213,668, filed Mar. 15, 1994).PCT/US/0307 describes a biocompatible, preferably biodegradablepolymeric matrix for containing an exogenous gene under the control ofan appropriate promoter. The polymeric matrix is used to achievesustained release of the exogenous gene in the patient. In accordancewith the instant invention, the agent of the invention is encapsulatedor dispersed within the biocompatible, preferably biodegradablepolymeric matrix disclosed in PCT/US/03307. The polymeric matrixpreferably is in the form of a micro particle such as a micro sphere(wherein the agent is dispersed throughout a solid polymeric matrix) ora microcapsule (wherein the agent is stored in the core of a polymericshell). Other forms of the polymeric matrix for containing the agents ofthe invention include films, coatings, gels, implants, and stents. Thesize and composition of the polymeric matrix device is selected toresult in favorable release kinetics in the tissue into which the matrixdevice is implanted. The size of the polymeric matrix devise further isselected according to the method of delivery which is to be used,typically injection into a tissue or administration of a suspension byaerosol into the nasal and/or pulmonary areas. The polymeric matrixcomposition can be selected to have both favorable degradation rates andalso to be formed of a material which is bioadhesive, to furtherincrease the effectiveness of transfer when the devise is administeredto a vascular surface. The matrix composition also can be selected notto degrade, but rather, to release by diffusion over an extended periodof time.

[0177] Both non-biodegradable and biodegradable polymeric matrices canbe used to deliver agents of the invention of the invention to thesubject. Biodegradable matrices are preferred. Such polymers may benatural or synthetic polymers. Synthetic polymers are preferred. Thepolymer is selected based on the period of time over which release isdesired, generally in the order of a few hours to a year or longer.Typically, release over a period ranging from between a few hours andthree to twelve months is most desirable. The polymer optionally is inthe form of a hydrogel that can absorb up to about 90% of its weight inwater and further, optionally is cross-linked with multi-valent ions orother polymers.

[0178] In general, the agents of the invention are delivered using thebioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone.

[0179] Examples of non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof.

[0180] Examples of biodegradable polymers include synthetic polymerssuch as polymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

[0181] Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein by reference, polyhyaluronic acids, casein, gelatin,glutin, polyanhydrides, polyacrylic acid, alginate, chitosan,poly(methyl methacrylates), poly(ethyl methacrylates),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(henyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate). Thus, the invention provides a composition of theabove-described agents for use as a medicament, methods for preparingthe medicament and methods for the sustained release of the medicamentin vivo.

[0182] The compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the therapeuticagents into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the therapeutic agent into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

[0183] Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the therapeutic agent, whichis preferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1, 3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Carrier formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. can be found inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa.

[0184] Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the therapeutic agent. Othercompositions include suspensions in aqueous liquors or non-aqueousliquids such as a syrup, an elixir, or an emulsion.

[0185] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the therapeutic agent of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, poly(lactide-glycolide), copolyoxalates,polyanhydrides, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polycaprolactone. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Nonpolymer systems that are lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-, di- and tri-glycerides; liposomes; phospholipids; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings,compressed tablets using conventional binders and excipients, partiallyfused implants and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the polysaccharide iscontained in a form within a matrix, found in U.S. Pat. Nos. 4,452,775,4,675,189, and 5,736,152, and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. Inaddition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

[0186] Use of a long-term sustained release implant may be particularlysuitable for treatment of established cancer conditions as well assubjects at risk of developing a cancer. “Long-term” release, as usedherein, means that the implant is constructed and arranged to delivertherapeutic levels of the active ingredient for at least 7 days, andpreferably 30-60 days. The implant may be positioned at the site of thetumor. Long-term sustained release implants are well known to those ofordinary skill in the art and include some of the release systemsdescribed above.

[0187] The therapeutic agent may be administered in alone or incombination with an anti-cancer drug. If the therapeutic agent isadministered in combination the compounds may be administered by thesame method, e.g. intravenous, oral, etc. or may be administeredseparately by different modes, e.g. therapeutic agent administeredorally, anti-cancer drug administered intravenously, etc. In oneembodiment of the invention the therapeutic agent and the anti-cancerdrug are co-administered intravenously. In another embodiment thetherapeutic agent and the anti-cancer drug are administered separately.

[0188] Anti-cancer drugs that can be co-administered with the compoundsof the invention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafir; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride. Additional antineoplastic agents include those disclosedin Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A.Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

[0189] Table 1: Peptide Sequences:

[0190] Histidine-Tagged Truncated Receptor (His-TR):

[0191] GTINVHDVETQFNQYKTEAASPYNLTISDVSVSHHHHHH (SEQ ID NO: 1)

[0192] Histidine-Tagged Primary Sequence of the MUC1 Growth FactorReceptor (His-PSMGFR):

[0193] GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH (SEQ ID NO:2)

[0194] Histidine-Tagged Extended Sequence of MUC1 Growth Factor Receptor(ESMGFR)

[0195] VQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVS DVPFPFHHHHHH (SEQ IDNO: 3)

[0196] Histidine-Tagged Primary Sequence of the Interchain bindingRegion (His-PSIBR):

[0197] HHHHHHGFLGLSNIKFRPGSVVVQLTLAFRE (SEQ ID NO: 4)

[0198] Histidine-Tagged Repeat Motif 2 (His-RM2):

[0199] PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAHHHHHH (SEQ ID NO: 5)

[0200] Truncated Receptor (TR):

[0201] GTINVHDVETQFNQYKTEAASPYNLTISDVSVS (SEQ ID NO: 6)

[0202] Primary Sequence of the MUC1 Growth Factor Receptor (PSMGFR):

[0203] GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO: 7)

[0204] Primary Sequence of the Interchain Binding Region) (PSIBR):

[0205] GFLGLSNIKFRPGSVVVQLTLAFRE (SEQ ID NO: 8)

[0206] Repeat Motif 2 (RM2):

[0207] PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 9)

[0208] MUC1 Receptor

[0209] (Mucin 1 Precursor, Genbank Accession Number: P15941

[0210] MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSVLSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTSAPDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTSASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHSSVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQIYKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVSVSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPARDTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL

[0211] (SEQ ID NO: 10)

[0212] Proopiomelanocortin(adrenocorticotropin/beta-lipotropin/alpha-melanocyte stimulatinghormone/beta-melanocyte stimulating hormone/beta-endorphin) [Homosapiens]. Accession number: XP_(—)002485

[0213] AAAKEGKKSR DRERPPSVPA LREQPPETEP QPAWKMPRSC CSRSGALLLA LLLQASMEVRGWCLESSQCQ DLTTESNLLE CIRACKPDLS AETPMFPGNG DEQPLTENPR KYVMGHFRWDRFGRRNSSSS GSSGAGQKRE DVSAGEDCGP LPEGGPEPRS DGAKPGPREG KRSYSMEHFRWGKPVGKKRR PVKVYPNGAE DESAEAFPLE FKRELTGQRL REGDGPDGPA DDGAGAQADLEHSLLVAAEK KDEGPYRMEH FRWGSPPKDK RYGGFMTSEK SQTPLVTLFK NAIIKNAYKK GE(SEQ ID NO: 11)

[0214] RGD

[0215] HHHHHHSSSSGSSSSGSSSSGGRGDSGRGDS (SEQ ID NO: 12)

[0216] The function and advantage of these and other embodiments of thepresent invention will be more fully understood from the examples below.The following examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.

EXAMPLES Colloid Preparation/Drug Screening Methods Employed in theExamples

[0217] In certain examples and embodiments of the invention, use is madeof self-assembled monolayers (SAMs) on surfaces of colloid particles.Colloids were derivatized with SAMs and prepared for drug screening in amanner similar to that described in International Patent Publication No.WO 00/43791, published Jul. 27, 2000, entitled “Rapid and SensitiveDetection of Aberrant Protein Aggregation in NeurodegenerativeDiseases”, incorporated herein by reference.

[0218] In a typical example, 1.5 ml of commercially available goldcolloid (Auro Dye by Amersham) were pelleted by centrifugation in amicrofuge on high for 10 minutes. The pellet was resuspended in 100 μLof the storage buffer (sodium citrate and tween-20). 100 μL of adimethyl formamide (DMF) solution containing thiols. Following a 3-hourincubation in the thiol solution, the colloids were pelleted and thesupernatant discarded. They were then heat cycled in 100 μL of 400 μMti-ethylene glycol-terminated thiol in DMF for 2 minutes at 55° C., 2minutes at 37° C., 1 minute at 55° C., 2 minutes at 37° C., then roomtemperature for 10 minutes. Heat cycling results in the elimination ofany species that are not in the lowest energy confirmation, resulting ina stable, close-packed, self-assembled monolayer. Heat cycling can becarried out with any of a wide variety of self-assembledmonolayer-forming species. The colloids were then pelleted and 100 μL100 mM NaCl phosphate buffer were added. The colloids were then diluted1:1 with 180 μM NiSO₄ in the colloid storage buffer.

[0219] Thiols used in coating colloids typically were derived fromsolutions containing about 40 μM nitrilo tri-acetic acid (NTA)-thiol,and other thiols such as methyl-terminated thiol (HS-(CH2)15 CH3), 40%tri-ethylene glycol-terminated thiol, HS(CH₂)₁₁(CH₂CH2)₃OH, (formula)and 50% poly (ethynylphenyl) thiol (C₁₆H₁₀S). Different thiols were usedto selectively inhibit non-specific binding optimally.

[0220] Colloid aggregation can be sensitively detected by monitoringcolor change of colloid particles which are initially disperse insuspension. Aggregation results in a color change to blue. No auxiliarysignaling entity is necessary. In drug screening, aggregation (or lackthereof) is observed in the presence of candidate drugs.

Example 1a Determination of Self-Aggregation of PSIBR Peptide of MUC1

[0221] The following experiment was designed to challenge the hypothesisproposed by others that a cleaved portion of the MUC1 receptor becomesthe ligand for that portion of the receptor that remains attached to thecell surface after shedding. However, surprising results indicate that aportion of the MUC1 receptor, close to the cell surface, self-aggregatesin a high affinity interaction, see FIG. 2. In FIG. 2, all wells of RowA, columns 1-4 and Column 1, rows A-D turned blue within a few minutes,while the remaining 9 wells (rows B-D, columns 2-4) remined pink. Thisexperiment tested various fragments of the MUC1 receptor for theirability to bind to each other or to themselves (self-aggregate). Resultsshow that none of the fragments bind to other portions of the receptor.However, a portion defined at least in part by the sequence of the PSIBRpeptide (Table 1) self-aggregates in a high affinity interaction. Wetermed this region the IBR (interchain binding region) of the receptor.

[0222] Histidine-tagged peptides were synthesized with the sequencesshown in table 1 (the various regions of MUC1). The lyophilized peptideswere dissolved in DMSO to give a final concentration of 5 mM. A stocksolution of each peptide was made by dissolving 4 μl 5 mM DMSO stock in196 μl Phosphate-Buffered-Saline (PBS) for a resulting concentration of100 μM. 20 μl of each 100 μM peptide solution was added to 100 μlcolloids presenting NTA-Ni to capture the histidine tag of the peptides.The colloids were incubated with the histidine-tagged peptides at roomtemperature for 10 minutes to allow binding of the histidine-tags to theNTA-Ni on the surface of the colloids. 20 μl aliquots of each peptidesspecies on colloids was then mixed with 20 μl of every other peptidespecies and with 60 μl of phosphate buffer pH 7.4. The color change ofthe colloid solutions was recorded after 15 minutes, see FIG. 2. Row Acontains the His-PSIBR (primary sequence interchain binding region)peptide; Row B contains the His-TR peptide; Row C contains the His-RM2peptide; Row D contains the His-PSMGFR peptide. Column 1 contains theHis-PSIBR peptide; Column 2 contains the His-TR peptide; Column 3contains the His-RM2 peptide; and Column 4 contains the His-PSMGFRpeptide. The solutions were observed for a color change. A change insolution color from pink to blue indicates that the colloids have beenforced together by a binding interaction. During this period, the set ofcolloids incubated with the His-PSIBR peptide changed color from pink toblue. Only the well of Row A, Column 1, which is the cross section ofPSIBR with itself, turned from pink to blue within the first 10 minutes.Results show that none of the receptor fragments bind to other portionsof the receptor, but importantly, one region which we term the primarysequence of the interchain binding region (PSIBR), self-aggregates in ahigh affinity interaction, suggesting a mechanism by which the MUC1receptor confers tumorigenesis.

[0223] No color changes were observed in wells that did not containPSIBR, indicating that the other peptide portions of MUC1 do notinteract with one another. However, after an hour-long incubationperiod, solutions that contained PSIBR and any other peptide, whichincluded MUC1-derived peptides as well as control peptides, turnedpurple, presumably due to self-aggregation of the PSIBR, which wassomewhat inhibited due to the presence of irrelevant peptides.

[0224] The colloid sets were centrifuged to form a pellet and wereresuspended in phosphate buffer. The PSIBR-peptide-bound colloids wouldnot resuspend in buffer, indicating that the binding interaction thathad forced the colloids together was a tight interaction. Since therewas only one peptide species on the colloids, the binding interactionmust be the PSIBR peptide binding to itself. The other sets of colloidsbound to peptides were assayed for their interaction with one another bymixing 15 μl of one colloid type with 15 μl of a second colloid type and70 μl phosphate buffer.

Example 1b Relationship Between MUC1 Cleavage Site in Tumor Conditionsand MUC1 Interchain Binding

[0225] This example investigates the ability of peptide sequences nearthe boundary between the MGFR and PSIBR of the MUC1 receptor toparticipate in self-aggregation, and thereby elucidates a probablecleavage site of MUC1 that is associated with tumorigenesis or cancer.

[0226] A histidine-tagged peptide (ESMGFR) whose sequence contained allof the amino acids in the His-PSMGFR peptide plus 9 additional aminoacids from the PSIBR region, adjacent to the PSMGFR, were added to theN-terminus of the peptide.N-terminus—VQLTLAFREGTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFHHHHHH—C-terminus.

[0227] The peptide was attached to colloid particles presenting NTAthiols, as in other experiments described herein. Thecolloid-immobilized ESMGFR peptides self-aggregated, which caused thecolloid solution to change color (from pink toward blue, see well B ofFIG. 3, which has changed from pink to blue). However, the extent ofcolor change, which indicates the extent of particle-immobilized peptideaggregation, was not nearly as dramatic as in identical experiments inwhich the colloid-immobilized His-PSIBR peptide was allowed toself-aggregate (well A of FIG. 3—blue), demonstrating someself-aggregation when a portion of the IBR region was attached to thenon-aggregating His-PSMGFR peptide. The His-PSMGFR peptide sequence wasdemonstrated to be completely free of self-aggregation. Specifically,the His-PSMGFR peptide was fastened to colloid particles and in anaggregation assay was shown not to self-aggregate (well C of FIG.3—pink).

[0228] This strongly suggests that cleavage of the MUC1 receptor intumors or cancers associated with aberrant expression of MUC1 occurs ator near the boundary between the PSMGFR and PSIBR sequences, since it isdemonstrated herein that in tumor cells that overexpress MUC1, the MGFRis accessible by agents that reduce cell proliferation by inhibiting theinteraction between MGFR and ligands that could be growth factors, andthat otherwise would promote cell proliferation. This also stronglysuggests that the IBR is shed in cleavage of MUC1 receptor in tumor orcancer associated with aberrant expression of MUC1, but is not shed incleavage of MUC1 when MUC1 is normally expressed in healthy cells. Thatis, that the cleavage site of MUC1 is at or near the C-terminal boundaryof the IBR in tumor or cancer cells and IBR at or near the N-terminalboundary of the IBR in healthy cells.

[0229] In the remaining examples, the mechanism described above forcancer associated with aberrant expression of MUC1, in which anactivating ligand (which is a growth factor) binds to multiple MGFRs ata cell surface and thereby triggers a signal within the cell whichcauses proliferation (inductive multimerization), is confirmed. Briefly,the mechanism is confirmed by showing that exposure of cells to abivalent antibody raised against MGFR induces cell proliferationcharacterized by a growth/response curve typical of a growthfactor/receptor-antibody response (Example 2, below); the activatingligand produced by MUC1-presenting cells binds multiple PSMGFRs, and theamount of activating ligand produced by each cell type is proportionalto the amount of MUC1 receptor produced by that cell type (Example 3a-b,below); MUC1 tumor cells produce a species that is a multimer (Example4b, below); and drugs found to be specific for MUC1 tumor cells (drugsthat inhibit proliferation in MUC1 tumor cells but not other cells) areshown to bind to MGFR at cells, while those that are not specific (thosethat inhibit MUC1 tumor cells and other cells) are toxic in that theybind to the multimeric ligand and thereby remove it from interactionwith the cells (Example 5b2, below).

Example 2 Dimerization of the MGFR Portion of the MUC1 Receptor TriggersEnhanced Cell Proliferation Consistent with the Mechanism Presented forMUC1 Tumor Cells

[0230] This example demonstrates the effect of dimerization on the MUC1receptor. In this example it is shown that exposure of cells to abivalent antibody grown against the MGFR region of the MUC1 receptor, atvarying concentration, results in enhanced cell proliferation (or lackthereof) consistent with the mechanism presented for MUC1 tumor cells. Abivalent antibody was raised against PSMGFR (i.e., a single antibodyhaving the ability to bind simultaneously to two MGFRs was produced).MUC1 tumor cells (T47Ds) were exposed to this antibody, and cellproliferation was studied as a function of concentration of theantibody. A growth/response curve typical of a growthfactor/receptor—antibody response was observed. Specifically, atconcentration low enough that only a small portion of the cells wereexposed to the antibody, cell proliferation was low. At a concentrationof antibody high enough that one antibody could bind adjacent MGFRs,cell proliferation was maximized. At a high excess of antibody, eachantibody bound only a single MGFR, rather than dimerizing adjacentMGFRs, and proliferation was reduced.

[0231] T47D (HTB-133) cells, a human breast cancer cell line thatoverexpresses MUC1, were cultured to 30% confluency. An antibody raisedagainst the PSMGFR portion of the MUC1 receptor, i.e. an antibody to theMFGR (Zymed, San Francisco, Calif., USA), was added to cells at varyingconcentrations in a multi-well cell culture plate. As a negativecontrol, a second set of T47D cells was treated with an irrelevantantibody (anti-streptavidin). Prior to adding antibody, cells werecounted (at time zero). All experiments were performed in triplicate.Cells were allowed to grow in a CO₂ incubator under normal conditions.Cells were counted using a hemacytometer (3 counts per well) at 24 hoursand again at 48 hours. Results, see FIG. 4, show that in aconcentration-dependent manner, addition of antibody caused enhancedcell proliferation compared to the proliferation of the same cellstreated with a control antibody. FIG. 4 is a graph in which measuredcell growth at 24 and 48 hours is plotted as a function of anti-PSMGFRconcentration. At the optimal antibody concentration, when presumablyone antibody binds bivalently to two MGFR portions of the MUC1 receptor,i.e. dimerizes the receptor, cell proliferation is at a maximum.

[0232] In a similar experiment, a concentration of the anti-PSMGFRantibody, identified to maximize cell proliferation, was added to afirst group of T47D tumor cells, grown as described above. The sameamount of the anti-PSMGFR antibody was added to a set of control cells,K293 cells. FIG. 5 shows that the addition of the anti-PSMGFR antibodyto MUC1 tumor cells (T47D) enhanced proliferation by 180% 24 hours, buthad no effect on the control cells. The growth of the T47D cellsplateaued to saturation, for cells with added antibody, at 48 hours.Control cells never reached saturation within the time frame of theexperiment and were at 70% confluency at 48 hours.

Example 3a The Activating Ligand Produced by MUC1-Presenting Cells BindsMultiple PSMGFRs

[0233] In this example, it is demonstrated that the activating ligandthat triggers MUC1 tumor cell proliferation binds multiple PSMGFRssimultaneously. Colloid particles were produced that carry immobilizedPSMGFRs, and suspensions of these colloids were exposed to lysate andsupernatants of (1) MUC1 tumor cells, or (2) control cells. MUC1 tumorcell lysates/supernatants caused the colloids to aggregate (suspensionturns blue) because the activating ligand contained in them binds MGFRson different colloid particle, causing the colloid particles toaggregate. The control cell lysates/supernatants do not.

[0234] 20 microliters of a 100 micro molar solution of the His-PSMGFRpeptide, described in Table 1, were added to 100 microliters of colloidswhich were derivatized with a SAM including NTA-Nickel moieties tocapture the histidine-tagged peptides. Lysates and supernatants fromfour different tumor-associated cell lines (HTB-133 (also called T47D),CRL-1500, CRL 1504 and CRL-1902; ATTC, American Type Culture Collection,Manasses, Va.) were added to aliquots of the peptide-presentingcolloids. To each well of a 96-well plate, 30 uL of eachlysate/supernatant was added to 40 uL of PBS, 30 ul of theHis-PSMGFR-bearing or as a negative control, the GST-bearing colloids. Acolor change from pink to blue rapidly occurred for the two cell linesthat overexpress the MUC1 receptor. A color change was observed for theCRL-1504 cells, which express MUC1, but after a much longer incubationperiod. No color change occurred for the cell line CRL-1902, which arenot known to express the MUC1 receptor. As a negative control, thelysates were also added to colloids presenting an irrelevant peptide,the GST protein. As an additional negative control (data not shown),growth media for the different cell lines was added to colloidpreparations. No color change was observed for the negative controls.

[0235] Results are shown in FIG. 6, which is an image of a section of a96-well plate illustrating the colloid-based color change assay.

[0236] Referring still to FIG. 6, wells in Column 1 contain colloidsbearing His-PSMGFR. Wells in Column 2 contain colloids bearing GST(glutathione-S-transferase) protein as a negative control. Row B:HTB-133 (also known as T47D) color change from pink to blue; Row C:CRL-1500 color change from pink to blue; Row A: CRL-1504 color changefrom pink to blue, but after a much longer incubation period; Row D:CRL-1902 no color change was produced. As a negative control, thelysate/supernatants were also added to colloids presenting an irrelevantpeptide, the GST protein, but no color change was observed for any cellline.

[0237] The results of FIG. 7 argue that this is a specific interactionbetween the PSMGFR peptide and a ligand(s) rather than randomprecipitation of colloids. FIG. 7 is an image of 96-well plateillustrating a colloid-based color-change binding assay between theHis-PSMGFR, MUC1-derived peptide and a ligand(s) present in a crude celllysate. The addition of imidazole, which releases the histidine-taggedprobe peptide from the NTA moiety on the colloid, caused a reversal ofthe color change, which argues that the color change is the result of aspecific interaction rather than random colloid aggregation. Addition ofa ligand present in a crude cell lysate of T47D cells, which may be adimer under these conditions, to the peptide-presenting colloidsolutions, caused the solution to turn blue, see FIG. 7 Row B, (atarrow). A drug candidate that disrupts the peptide-ligand interactionwill cause the solution to remain or revert to pink. In this way, highthroughput drug screening is achieved and may also be automated byanalyzing color change on a spectrophotometer.

[0238] These results indicate that cell lines HTB-133 (T47D) andCRL-1500 secrete high levels of the ligand for the MUC1 receptor andthat the ligand acts to dimerize or multimerize the receptor.

Example 3b Relationship Between Degree of Expression of MUC1 in CellLines and Presence of Ligand That Interacts With PSMGFR

[0239] In this example, it was shown that there is a direct correlationbetween the expression of MUC1 and the presence of a ligand or ligandsthat binds to a peptide derived from the portion of the MUC1 receptorthat is directly adjacent to the cell surface (MGFR).

[0240] Colloid particles carrying immobilized PSMGFR (His-PSMGFR linkedto NTA-presenting thiols in SAMs on the colloids) were observed fortheir aggregation potential (color change from pink to blue insuspension) upon exposure to lysates from various cells lines know tooverexpress, express, or not express MUC1. HTB-133, CRL-1500, CRL-1504,and CRL-1902 lines were studied. Lysates from a cell line thatoverexpresses MUC1 (HTB-133) caused colloid suspensions to turn bluewithin 15 minutes, indicating a high concentration of a ligand(s) in thelysate that interacts with the colloid-immobilized MGFR-derivedpeptides. Lysates from cell lines that express, but do notoverexpresses, MUC1 (CRL-1500 and CRL-1504) caused colloid suspensionsto turn blue within 3 hours, indicating moderate concentration ofligand(s) in the lysate. Lysates from a cell line that is not known toexpress MUC1 (CRL-1902) caused colloid suspensions to begin to turn blueonly after 10 hours, indicating a low concentration of a ligand(s) inthe lysate. Controls involved immobilized RGD peptide (which is notdimerized by components of these cell lysates). The control suspensionsremained pink indefinitely, indicating no aggregation.

[0241] See FIGS. 8A-15D. Rows A-D contained colloid particles carryingimmobilized His-PSMGFR. Rows E-H contained colloid particles carrying arandom sequence peptide. Columns 2, 5, 8, and 11 contained lysates froma tumor cell line that overexpresses MUC1 (HTB-133). Columns 3, 6, 9,and 12 contained lysates from a tumor cell line that does not expressesMUC1 (CRL-1902). Columns 1, 4, 7, and 10 contain lysates from a tumorcell line that expresses, but does not overexpress, MUC1 (CRL-1504).Columns 1-3: NTA concentration on colloid: 20 micromolar; columns 4-6:40 micromolar; columns 7-9: 60 micromolar; columns 10-12: 80 micromolar,all in total thiol concentration of 600 micromolar in depositionsolution. FIG. 8A: time=0; FIG. 8B: time=15 minutes; FIG. 8C: time=1hour; FIG. 8D: time=3 hours.

[0242] Overall, these results point to a mechanism involving a feedbackloop involving both ligand production and aberrant expression of theMUC1 receptor.

Example 4a Identification of Ligands that Bind to the MGFR Portion ofthe MUC1 Receptor

[0243] In an effort to identify ligands to the MUC1 receptor, synthetic,His-PSMGFR peptides, GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH(SEQ ID NO: 2), which represents the portion of the MUC1 receptor, thatremains attached to the cell surface after cleavage of the interchainbinding region, were loaded onto NTA-Ni beads (cat. #1000630; availablefrom Qiagen GmbH, Germany) and incubated with cell lysates in thepresence (FIG. 9) or absence (FIG. 10) of the protease inhibitor PMSF(phenyl methyl sulfonyl fluoride). Lysates from T47D cells were usedbecause this breast tumor cell line was known to overexpress MUC1;additionally, the inventors presented evidence herein (see FIGS. 8A-D)that this cell line also overexpresses MUC1 ligand(s). T47D cells werecultured then sonicated for 1 minute to lyse the cells. Lysates weremixed with the PSMGFR peptide-presenting beads and incubated on ice withintermittent mixing for 1 hr. As a negative control, an irrelevantpeptide, HHHHHHRGEFTGTYITAVT, was attached to NTA-Ni beads and treatedidentically. Both sets of beads were washed 2× with phosphate buffer pH7.4. Bound protein species were eluted by 3 additions of 100 uL ofphosphate buffer that also contained 250 mM imidazole. For both thepeptides, a portion of the first elution was removed and reserved to runas a separate sample, while the remainder was combined with the other 2elutions and concentrated by TCA (tri-chloro acetic acid)-precipitation(Chen, L. et al., Anal. Biochem. Vol 269; pgs 179-188; 1999). Eluateswere run on a 12% SDS gel, see FIG. 9. The gel was then silver stained(Schevchenko, A et al; Anal. Chem., Vol. 68; pg 850-858; 1996). Laneswere loaded as follows: (from left to right) (1) Benchmark pre-stainedprotein ladder (Gibco); (2) first eluate from the MUC1 peptide; (3){fraction (1/10)}^(th) of TCA-concentrated sample; (4) blank; (5){fraction (9/10)}^(th) TCA-concentrated sample; (6) first eluatenegative control peptide; (7) {fraction (1/10)}^(th) of TCA-concentratedsample from the negative control peptide; (8) 0.5 picomoles BSA (as astandard); (9) {fraction (9/10)}^(th) TCA-concentrated sample from thenegative control peptide; (10) silver stain SDS page standard (BioRadcat. #1610314). Referring now to FIG. 9, comparing lanes 2 and 6(control), it can be seen that the MUC1 PSMGFR peptide bounddistinguishably to three peptides: a first unique peptide that runs atan apparent molecular weight of 17 kD; and a second peptide (moreintense band) that runs at an apparent molecular weight of 23 kD. Notethat in lane 5, where the sample is the most concentrated, a thirdunique band is seen at about 35 kD.

[0244]FIG. 10 shows the results of an experiment, which was identical tothat shown in FIG. 9, with the exception that the protease inhibitorPMSF was not added. PMSF binds to and blocks the action of severalenzymes, such as proteases. This experiment was performed, in theabsence of PMSF, to determine whether an enzyme present in the lysatewas a ligand of the MUC1 receptor. Referring now to FIG. 10, comparinglanes 3 (control) and 7, it can be seen that the MUC1, PSMGFR peptidebound distinguishably to one peptide, with an apparent molecular weightof 35 kD. Note that this band was visible in FIG. 9 (with PMSF), but wasmuch fainter and only co-eluted from the most concentrated sample. Theseresults are consistent with the idea that the PFMGFR portion of the MUC1receptor is a substrate for a ligand of apparent molecular weight ofabout 35 kD and which may bean enzyme. As mentioned elsewhere herein,drug screens based on inhibition of binding between the PSMGFR and thisligand or the ligand in a crude cell lysate can identify compounds thatinhibit the action of this enzyme. TABLE 2 Cell lines were purchasedfrom the ATCC (American Type Culture Collection, Manasses, VA) and areall breast carcinoma cell lines. Some lines have been shown to expressor over express the tumor marker receptor MUC1, Her2/neu or theoncogenic enzyme cathepsin K. Gel Result Color change Expression of CellCo-elutes with assay - yes, species in cell Common ATCC line PSMGFRpeptide turned blue line name name 1. +++ ++++ Expresses MUC1 T-47DHTB-133 2. + − ND on MUC1 UACC-893 CRL- over expresses 1902 HER2/neu 3.+++ ++++ Overexpresses ZR-75-1 CRL- MUC1 1500 4. ++ + Express MUC1ZR-75-30 CRL- over express 1504 cathepsin K

[0245] TABLE 3 Cell line Growth Media HTB-133 RPMI 1640 media, purchasedfrom Mediatech supplemented with 1 mM sodium pyruvate, 10% FBS, 4.5 g/Lglucose and 1.5 g/L sodium bicarbonate, with 2 IU bovine insulin per mL.CRL-1902 Liebovitz L-15 media (Sigma), supplemented with 10% FBSCRL-1500 RPMI 1640 media from Mediatech supplemented with 1 mM sodiumpyruvate, 10% FBS, 4.5 g/L glucose and 1.5 g/L sodium bicarbonateCRL-1504 RPMI 1640 media from Mediatech supplemented with 1 mM sodiumpyruvate, 10% FBS

Example 4b Demonstration that the Ligand That Interacts with MUC1 CancerCells is a Multimer

[0246] In this example, it is demonstrated that a ligand produced byMUC1 cancer cells that triggers cell proliferation in these cells is amultimer.

[0247] Protein bands at 17 kD, 23 kD, and 35 kD were excised from thegels described above in Example 4a and submitted for peptide analysis.These gel bands purportedly contained ligands to the MGFR region of theMUC1 receptor. Recall that the 17 kD and 23 kD species bound to the MGFRpeptide in the presence of the protease inhibitor, PMSF, while the 35 kDspecies bound when PMSF was not added to the cell lysate mixture.

[0248] The following peptide analysis was performed. Samples derivedfrom the gel slices were proteolytically digested. Fragments were thenseparated by microcapillary HPLC which was directly coupled to anano-electrospray ionization source of an ion trap mass spectrometer.MS/MS spectra was obtained on-line. These fragmentation spectra werethen correlated to known sequences using the SEQUEST® algorithm inconjunction with other algorithms. Results were then manually reviewedto confirm consensus with sequences of known proteins.

[0249] Peptide sequences contained within both the 17 kD and the 23 kDbands (PMSF added to lysate) corresponded to a protein known asMetastasis Inhibition Factor NM23, which has been implicated in both thepromotion and inhibition of metastasis of human cancers. Whether therole of NM23 is a tumor supressor or promoter may depend on the type ofcancer. In ovarian, colon and neuroblastoma tumors, NM23 overexpressionhas been linked to a more malignant phenotype (Schneider J, Romero H,Ruiz R, Centeno M M, Rodriguez-Escudero F J, “NM23 expression inadvanced and borderline ovarian carcinoma”, Anticancer Res, 1996;16(3A): 1197-202). However, breast cancer studies indicate that reducedexpression of NM23 correlates with poor prognosis (Mao H, Liu H, Fu X,Fang Z, Abrams J, Worsham M J, “Loss of nm23 expression predicts distalmetastases and poorer survival for breast cancer”, Int J Oncol 2001March;18(3):587-91).

[0250] The sequences that were identified from the protein gel banddescribed in FIGS. 9 and 10 and that are derived from a proteinimplicated in many cancers called Metastasis Inhibition Factor NM23 areshown below in Table 4. NM23 exists as a hexamer and may recognize anunmodified form of the MGFR portion of the MUC1 receptor.

[0251] Peptide sequences that were identified from the 35 kD gel band(PMSF NOT added to lysate) corresponded to more than one proteinspecies, including 14-3-3, which is a signaling protein implicated inmany cancers, and cathepsin D, which is a protease and is alsoimplicated in tumor progression. 14-3-3 exists as a dimer and cansimultaneously bind to two, identical phospho-serine peptides. Thiswould dimerize the MGFR portion of the MUC1 receptor to trigger cellproliferation, which is consistent with the mechanism presented herein.Cathepsin D is a protease and may be involved in the cleavage of theMUC1 receptor.

[0252] The identity of these ligands is consistent with theMUC1-dependent cell proliferation mechanism that is disclosed herein,i.e., a ligand that dimerizes the MGFR portion of the MUC1 receptortriggers cell proliferation and cleavage of only a portion of the MUC1extracellular domain exposes the functional part of the receptor whichis defined by most or all of the PSMGFR sequence given in Table 1.

[0253] Consistent with methods of the invention, a therapeutic strategyis to identify compounds that either interrupt the interaction of one ofthe ligands with the MGFR portion of the MUC1 receptor, or to identifycompounds that bind to and block the action of the ligand(s). TABLE 4 17kD species identified herein from gel band 1) Metastasis InhibitionFactor NM23 gi: 127982 TFIAIKPDGVQR VM*LGETNPADSKPGTIR VMLGETNPADSKPGTIRNIIHGSDSVK GLVGEIIKR GLVGEIIK 23 kD species identified herein from gelband 1) Metastasis Inhibition Factor NM23 gi: 127982 TFIAIKPDGVQRYM*HSGPVVAM*VWEGLNVVK 35 kD identified herein from gel band 1) 14-3-3epsilon gi: 5803225 AAFDDAIAELDTLSEESYK AASDIAM*TELPPTHPIR YLAEFATGNDRDSTLIMQLLR YDEMVESMK VAGM*DVELTVEER HLIPAANTGESK 2) cathepsin D gi:4503143 DPDAQPGGELM*LGGTDSK DPDAQPGGELMLGGTDSK ISVNNVLPVFDNLM*QQKISVNNVLPVFDNLMQQK QPGITFIAAK 3) human annexin V with Prolinesubstitution by Thrionine gi: 3212603 GLGTDEESILTLLTSR DLLDDLKSELTGKSEIDLFNIR

Examples 5a-d Drug Studies Consistent with Mechanism Presented for MUC1Cancer

[0254] In these examples, drugs that inhibit proliferation in MUC1 tumorcells specifically were compared to drugs that inhibit proliferation inboth MUC1 tumor cells and other cells. Drugs, both specific andnon-specific, were identified by exposing them to PSMGFR-presentingcolloids in the presence of MUC1 tumor cell lysates. Drugs wereidentified as those that prevented colloid-colloid interactions. Cellstudies resulted in a separation of these drugs into two groups—a groupspecific for MUC1 tumor cells and a non-specific group. Non-specificdrugs did not bind to PSMGFR, but are presumed to bind the activatingligand, and inhibit proliferation of control cells as well asMUC1-presenting cells. Additionally, this group of drugs was somewhattoxic to both cell types, since they remove the activating ligand frominteraction with the cells. Drugs specific for MUC1 tumor cells werefound to bind to PSMGFR on beads, as demonstrated by HPLC analysis ofthe product of cleavage of PSMGFR from the beads.

Example 5a Drug Screening Assay Using Colloid-Based ColorimetricDetection to Identify Agents that Block the Interaction of the MGFRPortion of the MUC1 Receptor with its Activating Ligand(s)

[0255] The following is an example of a working drug screening assay toidentify anti-cancer agents. In this example, a histidine-tagged peptidederived from the portion of the MUC1 receptor that remains attached tothe cell surface after receptor cleavage (His-PSMGFR) was attached toNTA-nickel-SAM-coated gold colloids. The peptide-presenting colloidswere incubated with lysates/supernatants from MUC1 presenting cells thatwere shown herein to contain ligands that cause dimerization ormultimerization of that portion of the MUC1 receptor. Thisligand-induced multimerization of the MGFR portion of the receptorcauses the attached colloids to be drawn close together, which causes achange in the color of the colloid solution from pink to blue. Drugsthat interfere with the binding of activating ligands to the MGFRportion of the receptor cause the solution to remain pink.

[0256] NTA-SAM-coated colloids presenting the PSMGFR peptide wereincubated with cell lysates/supematants from T47D cells, which wepreviously showed by gel-electrophoresis to contain the ligand to MUC1(Example 4a). Negative control wells contained colloids bound with arandom sequence histidine-tagged peptide in place of the MUC1 peptide.FIG. 11, which is an image of 96-well plate illustrating a color-changebinding assay, shows the results of the experiment. The well containingcolloids that presented the PSMGFR peptide plus T47D cell lysates (WellA) changed color from pink to blue, indicating the presence of amultimerizing ligand. Wells that contained a random sequence peptide(RGD) in place of the PSMGFR peptide (Well B) remained pink. Wells thatcontained phosphate buffer in place of cell lysate also remained pink(Wells C and D).

[0257] The data below demonstrates the ability of anti-tumor drugsidentified in accordance with the invention, specifically, calcimycin,fusaric acid, L-α-methyl-dopa, butylindazone, NS1619 and etomoxir toinhibit proliferation of cells that aberrantly express MUC1, by blockingthe interaction of MGFR with ligands that promote cell proliferation.

[0258] An experiment similar to that described above was run in thepresence of drug candidates to determine whether candidates could beidentified that interfere with ligand/MGFR binding.

[0259] T47D cells were trypsinized from a T25 flask, pelleted,resuspended in phosphate buffer, and lysed by sonication to release theligand into solution. NTA-SAM-coated colloids were bound with theHis-PSMGFR peptide: GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGAHHHHHH200 μl NTA-SAM-coated colloids were incubated with 20 μl 100 μM peptidein phosphate buffer for 15 minutes, pelleted to remove unbound peptide,and resuspended in phosphate buffer. Negative control colloids wereincubated with a random sequence histidine-tagged peptide in place ofthe MUC1 peptide. The cell lysate (65 μl) was mixed with 5 μl drugcandidate in DMSO and added to 30 μl MUC1-peptide-bound colloids in thewells of an ELISA plate for a final drug concentration of approximately100 μM. Positive controls contained DMSO in place of a drug candidate;negative controls contained DMSO in place of a drug candidate, andcolloids bound with a random sequence peptide in place of the MUC1peptide. A color change from pink to blue indicates that the ligand inthe cell lysate bound to the MUC1-peptide, dimerizing the peptide, andbringing the colloids into close enough proximity with one another tocause a color change. Positive controls, which do not contain a drugcandidate, change color from pink to blue within two hours, as there isnothing to inhibit the interaction between the MUC1 peptide and theligand present in the cell lysate. A lack of color change (wells remainpink) indicates that the drug candidate blocked the interaction betweenthe MUC1 peptide and the cognate ligand, either by binding to the MGFRportion of the MUC1 receptor, inhibiting a modifying enzyme, or bybinding to its activating ligand. Negative control wells, which containcolloids presenting a random sequence peptide in place of the MUC1peptide, remain pink, as the ligand to the MUC1 peptide will notdimerize the random sequence peptide. FIG. 12 shows a sampledrug-screening plate used in the assay described above. Positive controlwells (A1-D1) changed color from pink to blue within two hours, whilenegative control wells (E1-H1) remained pink. Well E6 contains a drugthat inhibited the interaction between the MUC1 peptide and the cognateligand, causing the well to remain pink.

Example 5b1 Secondary Drug Screen to Determine Mode of Action ofIdentified Drug Agents—Proliferation of Cells Treated With DrugCandidates That Disrupt the Interaction of MUC1 With its Natural Ligand

[0260] Previously, we showed in vitro that the ligand to the MGFRportion of the MUC1 receptor caused dimerization, or multimerization ofthe PSMGFR peptide on colloids, see Example 3a. We also showed thatdimerization of the MGFR portion of the receptor induced an enhancedcell proliferation, see Example 2. It then follows that agents thatblock the interaction of the MGFR portion of the receptor with itsactivating ligands will block the proliferation of MUC1-presenting tumorcells. Therefore, drugs that were identified using the in vitro drugscreening assay described in Example 5a were tested in a functionalassay to determine their ability to inhibit MUC1-dependent cellproliferation.

[0261] T47D cells, mammary carcinoma cells known to overexpress MUC1,were grown in the wells of an ELISA plate along with K293 cells, humanembryonic kidney cells which will serve as the negative control. 100 μlcells in growth media were added to the wells of an ELISA plate and thecells were allowed to adhere overnight. The number of cells in each wellon the plate were then counted and recorded. 1 μl of a drug candidate inDMSO was then added to each well of both the T47D cells and the K293cells. 1 μl DMSO alone was added to control wells. Each well wasrepeated in triplet. The cells were allowed to grow for 48 hours, thenormal doubling time for these cell lines. The number of cells in eachwell was again counted and recorded. The percent cell growth over this48-hour period was calculated, and the percent cell growth for wellscontaining a drug candidate versus DMSO were compared. As seen in FIG.13, Etomoxir, L-alpha-methyl DOPA, and Fusaric acid selectivelyinhibited proliferation of the MUC1-expressing tumor cells over K293negative control cells. The DMSO control cells (both T47D and K293) showthat DMSO alone does not effect cell proliferation. FIG. 13 is ahistogram illustrating the selective inhibition of proliferation oftumor cells that aberrantly express the MUC1 receptor (T47D cell line),in response to treatment with compounds of the invention, and lack of aneffect on cells that do not express MUC1 (K293). Cell growth in thepresence of Etomoxir was −10.2% for T47D cells and 94% for control cells(K293); Cell growth in the presence of L-alpha-methyl-DOPA was 20% forT47D cells and 110% for control cells (K293); Cell growth in thepresence of Fusaric acid was 27.7% for T47D cells, and 106.7% forcontrol cells (K293); Cell growth in the absence of any drug (but withan equivalent amount of DMSO added) was 91.3% for T47D cells, and 106.2%for control cells (K293).

Example 5b2 Drugs Identified with the Colorimetric in vitro Drug ScreenSeparate into Two Categories—Selective for MUC1-Presenting Cells andNon-Selective

[0262] As discussed, compounds identified in the in vitro drug screeningassay (described in Example 5a), which identifies compounds thatinterfere with the interaction between the MGFR portion of the MUC1receptor and its activating ligands, can inhibit cell proliferation bythree modes of action. These drugs can a) block the activity of theactivating ligand(s), that act as growth factors; b) directly bind toand block the MGFR portion of the receptor; or c) inhibit the activityof enzymes that modify the MUC1 receptor. It is expected that drugs thatfunction by (a) will inhibit proliferation of a variety of cell types,while those that function according to (b) and (c) will selectivelyinhibit the proliferation of MUC1-presenting cells.

[0263]FIG. 14 is an image of a multi-well plate in which thecalorimetric drug screening assay (see Example 5a) identified severalcompounds (each designated by a MN#) that interfered with theinteraction of the MGFR portion of the MUC1 receptor and a multimerizingligand(s). All of the drug-containing wells demonstrate interferencewith ligand binding as evidenced by each of the wells either remainingpink or turning purple, indicative of binding being essentiallyeliminated or reduced over positive binding controls (top three wells ofright-most column, which are blue). Wells in the top half of the plate(rows A-C) contain drugs that were shown in the functional cellproliferation assay (see Example 5b) to selectively inhibit theproliferation of MUC1-presenting tumor cells by either directly bindingto the MGFR portion or by acting on its modifying enzymes. FIG. 15 is abar graph that compares the percentage cell growth of MUC1 tumor cells(T47Ds) to a control cell line (K293s), in response to treatment withnovel drugs, (described in greater detail in commonly-owned, co-pendingU.S. provisional patent applications serial Nos. 60/317,302 and60/317,314, both filed on Sep. 5, 2001 and entitled COMPOSITIONS ANDMETHODS OF TREATMENT OF CANCER). As is readily apparent, this group ofdrugs dramatically inhibited or completely prevented the proliferationof MUC1-presenting tumor cells, while leaving the control cells, in mostcases, unaffected.

[0264] Wells in the bottom half of the plate (rows D-G) contain drugsthat were shown in the cell proliferation assay to act non-selectivelyas they inhibited the proliferation of both cell types. FIG. 16 is a bargraph that shows the effect of these novel drugs (described in greaterdetail in commonly-owned, co-pending U.S. provisional patent applicationserial Nos. 60/317,302 and 60/317,314, both filed on Sep. 5, 2001 andentitled COMPOSITIONS AND METHODS OF TREATMENT OF CANCER) on cell growthfor MUC1-presenting cells (T47D) and a control cell line (K293).Notably, this group of drugs, which presumably bind to growth factors,is toxic to the cells as one skilled in the art would expect for agentsthat act on growth factors.

Example 5c Co-Elution of Drugs With PSMGFR Peptide Proves Direct Bindingto MGFR

[0265] Herein, we show that several drugs inhibited MUC1-dependent cellproliferation by binding to the MGFR portion of the MUC1 receptor anddisrupting the interaction between the MGFR portion and its cognateligand. The direct binding of several drugs to the PSMGFR peptide wasdemonstrated using two methods. The His-PSMGFR peptide was attached toNTA-nickel agarose beads then separately incubated with each of the drugcandidates. 100 μl beads were bound to saturation with 1 mg peptide,rinsed to remove unbound peptide, and incubated with 25 μl 2.7 mg/mldrug candidate in DMSO for one hour in 5 ml phosphate buffer. Unbounddrugs were washed away with phosphate buffer, and the peptide was elutedfrom the beads with PBS, 250 mM imidazole. If the drug candidate boundto the peptide, it would co-elute with the peptide in the imidazolesolution. Peptide-drug complexes were then separated by HPLC. HPLCelution peaks from the complex were compared to the elution peaks fromthe drug, injected alone, and the PSMGFR peptide alone. In a pilotstudy, 3 drugs, chosen randomly from the group of drugs that selectivelyinhibit MUC1 cell proliferation, were compared to 3 drugs chosenrandomly from the group that non-selectively inhibited cellproliferation. As seen in FIG. 17, the drugs on the left, chosen fromthe selective group, bind to the PSMGFR peptide, while drugs on theright, chosen from the non-selective group, did not.

[0266] In a similar experiment, eluates from the beads were analyzed byTLC (thin layer chromatography). Two drugs, calcimycin and NS1619 whichcould be tracked by TLC, because they fluoresced under UV light, weretested. NTA-nickel bead-immobilized peptides were incubated with thedrugs as described above, rinsed and eluted from the beads, thenrotoevaporated to remove the aqueous buffer. The solid was thenresuspended in ethylacetate, 5% methanol and spotted on TLC plates thenrun in the same organic solvent. Both drugs co-eluted with the PSMGFRpeptide, showing conclusively that the drugs bind to the PSMGFR peptide.Calcimycin, gave a clear blue spot under 250 nm UV light and ran at aless polar position (as expected) than the peptide itself. The seconddrug, NS1619, gave a less visible spot at a slightly less polar positionthan the peptide. The TLC plate was stained with iodine to reveal thepeptide spots, which are visible due to the presence of tyrosine andphenylalanine residues.

Prophetic Example 5d Discriminating between Drugs that InhibitMUC1-Dependent Cell Proliferation by Binding to and Blocking the MGFRPortion of the MUC1 Receptor and Those that Inihibit SelectiveProliferation by Acting on Enzymes that Modify the MGFR Portion

[0267] This experiment is designed to distinguish drugs that selectivelyinhibit MUC1 cell proliferation by inhibiting modifying enzymes fromdrugs that inhibit by binding to and blocking the MGFR portion of theMUC1 receptor. Ex. 8 below shows that ligand(s) present in the MUC1lysate were not able to bind PSMGFR peptide when the enzyme inhibitorPMSF was added to the lysate. This implied that the PSMGFR is firstmodified before it recognizes its cognate ligand(s). Using the drugscreening assay described in Ex. 5a, one cannot differentiate betweendrugs that will selectively block the proliferation of MUC1-presentingcells and those that block the proliferation of a wide variety of cells,i.e. by inhibiting the MUC1 ligands that act as growth factors. Toidentify drugs that are selective for MUC1, drug hits are subjected to asecondary assay, which measures the percentage cell proliferation ofMUC1 cells compared to control cells. Of the drugs that are selectivefor MUC1-presenting cells, neither assay can differentiate between drugsthat bind to and block the MGFR and those that inhibit its modifyingenzymes. Since enzyme modified peptides migrate through a gel at aslower rate than unmodified peptides, one can use this difference in gelmobility to determine which drugs act by inhibiting the enzyme(s).

[0268] To determine which drugs function via which of these twomechanisms, drugs are incubated with lysates and supernatants fromMUC1-presenting cells, to allow the drugs to inhibit MUC1-modifyingenzymes. The synthetic His-PSMGFR peptide, immobilized on commerciallyavailable NTA-nickel beads, are then mixed with this lysate mixture.After a 30-minute incubation on ice, excess lysate and drug arediscarded in the supernatant after the beads and attached peptide arepelleted by centrifugation. Peptides are then released from the beads bythe addition of 100 μL of 25 mM of imidazole. Samples are then analyzedby standard methods of SDS-PAGE on a 15% polyacrylimide gel. Theunmodified PSMGFR peptide runs with an apparent MW of 9 kd, while themodified peptide runs at an apparent MW of 11 kd. A shift to the MWcharacteristic of the unmodified peptide, after incubation with lysateand drug candidate, indicates that the drug under study acts byinhibiting a modifying enzyme.

Example 6 Modulation of Inhibitory Effect of Etomoxir on CellProliferation

[0269] Etomoxir, identified as a composition useful in treatment ofMUC1-dependant tumors in this invention, was shown to be specific forMGFR by modulating its effect on cell proliferation via competetiveinhibition of the MGFR/drug interaction by adding excess PSMGFR in cellgrowth media.

[0270] The following experiment was performed in triplicate. T47D cellswere grown to approximately 30% confluency. Etomoxir (approx. 100micromolar) was added and cell proliferation was observed to bearrested. Then, a synthetic peptide (PSMGFR) was added to the cellgrowth media under normal cell growth conditions. Addition of PSMGFRcaused increased cell proliferation, due to consumption of Etomoxir byPSMGFR (curve A of FIG. 18). As a control, cells were exposed toEtomoxir and a control peptide (RGD) of approximately the same molecularweight as PSMGFR and cell proliferation (curve B of FIG. 18) did notincrease to the extent that occurred when PSMGFR was added.

Example 7 Evidence for Enzyme Modification of the MGFR Portion of theMUC1 Receptor

[0271] The following experiment was performed to investigate thepossibility that the portion of the MUC1 receptor that remains attachedto the cell surface after receptor cleavage (MGFR) is enzyme-modified.Synthetic, His-PSMGFR peptides, (SEQ ID NO: 2) were loaded onto NTA-Nibeads and either incubated with T47D cell lysates or as a negativecontrol, incubated with cell growth media. Incubation was for 1 hour, onice with intermittent mixing. As a second negative control, anirrelevant peptide, RGD (SEQ ID NO: 12) was attached to NTA-Ni beads andtreated identically. Both sets of beads were washed 2× with phosphatebuffer pH 7.4. Bound protein species were eluted by addition of 250 mMimidazole. Lanes were loaded as follows: (from right to left) (10)pre-stained protein ladder (Gibco); (9) bead immobilized PFMGFR peptidewhich was not incubated with cell lysates; (8-6) eluates of beadimmobilized PFMGFR peptide which was incubated with T47D cell lysates;(5) eluates of bead immobilized negative control peptide which was notincubated with T47D cell lysate; (4-2) eluates of the negative controlpeptide was incubated with the lysate. Referring now to FIG. 19,comparing lanes 6-8 and 9 (control), it can be seen that afterincubation with the lysates (6-8) the MUC1 PSMGFR peptide runs at ahigher molecular weight than when it was incubated with cell growthmedia (9). Comparing lanes 5 and 2-4, there is no change in the apparentmolecular weight of the control peptide after incubation with the celllysate. These results are consistent with the idea that a ligand in thelysate of T47D cells is modifying the PSMGFR peptide portion of the MUC1receptor.

[0272] These results are also consistent with the idea that the ligandis an enzyme that covalently couples two adjacent MGFR portions of theMUC1 receptor, causing dimerization of the receptors and initiates acell proliferation signaling cascade. According to this mechanism, drugsof the invention interrupt cell proliferation by inhibiting the actionof this enzyme.

Example 8 Demonstration that the MGFR Peptide is Enzymatically ModifiedBefore its Natural Ligands Recognize It

[0273] To test the hypothesis that the MGFR portion of the MUC1 receptoris enzymatically modified prior to binding to its ligand(s), the drugscreening assay described in Example 5a was performed in the presence orabsence of an enzyme inhibitor, PMSF (phenylmethylsulfonyl fluoride;Sigma chemical Co. St. Louis Mo., USA). T47D cells, which are breasttumor cells that aberrantly express MUC1, were grown to 70% confluency,treated with trypsin to detach from the flask, and pelleted bycentrifugation. A lysate was prepared as follows from the pellets of twoT75 flasks. Cell pellets were resuspended in 300 μL phosphate buffer (10mM sodium phosphate, 100 mM NaCl, pH 7.4). The lysate was divided intotwo parts. One aliquot of lysate was used as is. To the second aliquotwas added 9 μL of a 100 mM stock solution to achieve a finalconcentration 3 mM. The lysates were frozen and thawed four times with15 seconds of vigorous mixing after each thawing. Lysates were thenpelleted by centrifugation and the supernatant collected. Lysates werediluted by adding 6.2 ml of phosphate buffer. The drug screening assaywas then performed as described in Example 5a, with the exception thatno drugs were added. Rather the ability of the ligand(s) in the lysateto bind to the MGFR in a multimeric way, which would result in thesolution color to change to blue, was tested. As can be seen in FIG. 20,solutions containing PMSF did not change color and remained pink. WellsA 1&2 turned from pink to blue within an hour and are the positivecontrol wells, which contain the His-PSMGFR peptide immobilized on goldcolloids and lysates/supernatants from T47D cells. Wells A 3&4 containthe same components as wells A 1&2, with the exception that thelysate/supernatant mixture was first treated with the enzyme inhibitorPMSF; wells containing PMSF do not undergo the solution color change andremain pink. Wells B 1&2 are negative control wells that contain thecolloid-immobilized His-PSMGFR peptide but are incubated with bufferrather than lysates, and remain pink. Wells C 1&2 are also negativecontrol wells in which the peptide immobilized on the colloids is anirrelevant peptide (RGD) that is incubated with the lysate, and theyremain pink as well.

[0274] Prophetic Example Involving Screening for Drugs That Affect MUC1Cleavage State

[0275] The release of the MUC1 IBR can be correlated to the progressionof cancer. The following is a description of a whole cell assay thatidentifies drug candidates that affect cleavage state of thesereceptors. The screen also identifies drug candidates that directly orindirectly modulate any step, including but not limited to enzymecleavage, receptor production, expression, stability, transport orsecretion, that ultimately results in a reduction of theself-aggregating portion of the receptor being shed and released fromthe cell.

[0276] Tumor derived cells expressing a cell surface receptor of thetype described above, are cultured and treated with a drug candidate.Following some incubation period, a peptide aggregation assay isperformed on the solution surrounding the cell. Colloids bearing abinding peptide e.g. an antibody against a constant region of thereceptor, remote from the enzyme cleavage site (amino acid 425-479 forMUC1; numbers refer to Andrew Spicer et al., J. Biol. Chem Vol 266 No.23, 1991 pgs. 15099-15109; these amino acid numbers correspond tonumbers 985-1039 of Genbank accession number P15941; PID G547937), areadded to the solution. If the shed portion of the receptor contains theself-aggregating portion, the receptors in solution will aggregate andcause the attached colloids to aggregate, causing a visibly detectablechange in the solution, for example: color change or the formation ofvisible aggregates. An inhibition of this visible change indicates anagent that is effective for treating the disease state.

[0277] The list of sequences in Table 1 is representative of sequencefragments that are found within the overall sequence of the full-lengthpeptide. Any set of at least 10 contiguous amino acids within any of thesequence fragments of Table 1 may be sufficient to identify the cognatebinding motif. The list of sequences of Table 1 is meant to embrace eachsingle sequence and when mentioning fragment size, it is intended that arange embrace the smallest fragment mentioned to the full-length of thesequence (less one amino acid so that it is a fragment), each and everyfragment length intended as if specifically enumerated. Thus, if afragment could be between 10 and 15 in length, it is explicitly meant tomean 10, 11, 12, 13, 14, or 15 in length.

[0278] With reference to Table 1, the receptor can be cleaved at anumber of different sites to generate peptide fragments with alternativebeginnings and endings. For these fragments of Table 1 any stretch of 8to 10 contiguous amino acids, either upstream or downstream, may beenough to identify the particular fragment that is the binding entityreferred to herein.

[0279] In addition to the diagnostics and screening assays of theinvention, the invention relates to therapeutic methods for thetreatment and prevention of cancer and related products. For instance,in one aspect the invention relates to a method for treating a subjecthaving a cancer or at risk of developing cancer by administering to thesubject an agent that reduces cleavage of a cell surface receptor IBRfrom a cell surface receptor.

[0280] Those skilled in the art would readily appreciate that allparameters listed herein are meant to be exemplary and that actualparameters will depend upon the specific application for which themethods and apparatus of the present invention are used. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, the invention may be practiced otherwise thanas specifically described. Specifically, those of ordinary skill in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

[0281] Several methods are disclosed herein of administering a subjectwith a compound for prevention or treatment of a particular condition.It is to be understood that in each such aspect of the invention, theinvention specifically includes, also, the compound for use in thetreatment or prevention of that particular condition, as well as use ofthe compound for the manufacture of a medicament for the treatment orprevention of that particular condition.

[0282] In the claims, all transitional phrases such as “comprising”,“including”, “carrying”, “having”, “containing”, “involving”, and thelike are to be understood to be open-ended, i.e. to mean including butnot limited to. Only the transitional phrases “consisting of” and“consisting essentially of”, respectively, shall be closed orsemi-closed transitional phrases as set forth in the United StatesPatent Office Manual of Patent Examining Procedures, section 2111.03.

[0283] In the claims, amino acid sequence numbers are as listed inAndrew Spicer et al., J. Biol. Chem Vol 266 No. 23, 1991 pgs.15099-15109.

We claim:
 1. A kit comprising: a first article having a surface; apeptide sequence immobilized relative to or adapted to be immobilizedrelative to the surface, the peptide sequence including a portion of acell surface receptor that interacts with an activating ligand such as agrowth factor to promote cell proliferation, the portion includingenough of the cell surface receptor to interact with the activatingligand and the portion free of interchain binding region to the extentnecessary to prevent spontaneous binding between portions; and acandidate drug for affecting the ability of the peptide sequence to bindto other identical peptide sequences in the presence of the activatingligand.
 2. A kit as in claim 1, further comprising a second articlehaving a surface and the peptide sequence immobilized relative to oradapted to be immobilized relative to the surface of the second article.3. A kit as in claim 1, wherein the peptide sequence is MGFR.
 4. Amethod comprising: providing a peptide including a portion of a cellsurface receptor that interacts with an activating ligand such as agrowth factor to promote cell proliferation, the portion includingenough of the cell surface receptor to interact with the activatingligand and the portion free of interchain binding region to the extentnecessary to prevent spontaneous binding between portions; exposing thepeptide to a candidate drug for affecting the ability of the activatingligand to interact with the peptide, and to the activating ligand; anddetermining the ability of the candidate drug to prevent interaction ofthe activating ligand with the peptide.
 5. A method as in claim 4,comprising determining the ability of the candidate drug to preventinteraction of the peptide with other proteins or peptides.
 6. A methodas in claim 4, comprising providing a first article having a surface anda plurality of the peptides immobilized relative to or adapted to beimmobilized relative to the surface; exposing the peptides and thesurface of the first article to the candidate drug and at least oneactivating ligand; and determining the ability of the candidate drug toprevent interaction of the activating ligand with the peptide.
 7. Amethod as in claim 4, comprising providing a first article having asurface, a second article having a surface, and a plurality of thepeptides immobilized relative to or adapted to be immobilized relativeto the surfaces of the first and second articles; exposing the peptidesand the surfaces of the first and second articles to the candidate drugand at least one activating ligand; and determining immobilization ofthe first and second articles relative to each other.
 8. A method as inclaim 4, wherein the step of exposing the peptides to the candidate drugand at least one activating ligand comprises exposing the peptide andthe candidate drug to one or both of cell lysate and cell supernatantcontaining the activating ligand.
 9. A method as in claim 4, wherein thepeptide sequence is PSMGFR.
 10. A method of treating a subject to reducethe risk of or progression of cancer comprising: administering to asubject who is known to be at risk for cancer or is diagnosed withcancer an agent for inhibiting interaction of an activating ligand witha portion of a cell surface receptor that interacts with the activatingligand to promote cell proliferation.
 11. A method as in claim 10,comprising administering to the subject an agent for inhibitinginductive multimerization of the portion of the cell surface receptorthat interacts with the activating ligand to promote cell proliferation.12. A method as in claim 10, wherein the cell surface receptor is MUC113. A method as in claim 10, wherein the portion of the cell surfacereceptor is MGFR.
 14. A method as in claim 10, wherein the portion ofthe cell surface receptor contains a significant part of the PSMGFRsequence.
 15. A method as in claim 10, comprising administering to thesubject an agent for inhibiting dimerization of the portion of the cellsurface receptor that interacts with the activating ligand to promotecell proliferation.
 16. The method of claim 10, wherein the cancers isselected from the group consisting of: breast, prostate, lung ovarian,colorectal, and brain cancer.
 17. A method as in claim 10, wherein theactivating ligand is a multimer.
 18. The method of claim 10, wherein theactivating ligand is a protein with a molecular weight of about 17 kD.19. The method of claim 10, wherein the activating ligand is a proteinwith a molecular weight of about 23 kD.
 20. The method of claim 10,wherein the activating ligand is a protein with a molecular weight ofabout 35 kD.
 21. The method of claim 10, wherein the activating ligandcontains sequences derived from the protein 14-3-3.
 22. The method ofclaim 10, wherein the activating ligand contains sequences derived fromcathepsin D.
 23. The method of claim 10, wherein the activating ligandcontains sequences derived from NM23.
 24. The method of claim 10,wherein the activating ligand contains sequences derived from humanannexin V.
 25. The method of claim 10, wherein the activating ligandcontains sequences derived from beta-lipotropin.
 26. The method of claim10, wherein the activating ligand is a cleavage product ofproopiomelanocortin.
 27. The method of claim 10, wherein the portion ofthe cell surface comprises at least 12 contiguous amino acids from thesequence GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA.
 28. The methodof claim 10, wherein the portion of the cell surface receptor thatremains attached to the cell surface after shedding of the cell surfacereceptor interchain binding region comprises at least 12 contiguousamino acids from the peptide sequence GTINVHDVETQFNQYKTEAASPYNLTISDVSVS.29. The method of claim 10, wherein the agent is selected for use in themethod by determining its ability to bind to a significant portion ofthe peptide, GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA.
 30. Themethod of claim 10, wherein the agent is selected for use in the methodby determining its ability to bind to a significant portion of thepeptide sequence GTINVHDVETQFNQYKTEAASPYNLTISDVSVS.
 31. A method oftreating a subject to reduce the risk or of progression of cancercomprising: administering to a subject who is known to be at risk ofcancer or is diagnosed with cancer, an agent for preventative clusteringof portions of cell surface receptors that interact with an activatingligand such as a growth factor to promote cell proliferation.
 32. Themethod of claim 31, wherein the cell surface receptor is MUC1.
 33. Amethod as in claim 31, comprising contacting the portions of the cellsurface receptor with a molecule that can bind to multiple portionsthereby clustering a plurality of the portions.
 34. A method as in claim31, wherein the portion is MGFR
 35. A method as in claim 31, wherein theportion contains a significant amount of the PFMGFR sequence.
 36. Themethod of claim 31, wherein the cancer is selected from the groupconsisting of: breast, prostate, lung ovarian, colorectal, and braincancer.
 37. The method of claim 31, wherein the portion of the cellsurface receptor comprises at least 12 contiguous amino acids from thepeptide sequence GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA.
 38. Themethod of claim 31, wherein the portion of the cell surface receptorcomprises at least 12 contiguous amino acids from the peptide sequenceGTINVHDVETQFNQYKTEAASPYNLTISDVSVS.
 39. The method of claim 31, whereinthe specific binding portion of the agent is selected for use in themethod by determining its ability to bind to a significant portion ofthe peptide, GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA.
 40. Themethod of claim 31, wherein the specific binding portion of the agent isselected for use in the method by determining its ability to bind to asignificant portion of the peptide, GTINVHDVETQFNQYKTEAASPYNLTISDVSVS.41. A kit comprising: a species able to become immobilized relative to ashed cell surface receptor interchain binding region; and a signalingentity immobilized relative to or adapted to be immobilized relative tothe species.
 42. A kit as in claim 41, wherein the species binds to aportion of a shed MUC1 receptor that is connected to the interchainbinding region.
 43. A kit as in claim 41, wherein the cell surfacereceptor is MUC1.
 44. The kit as in claim 41, wherein the signalingentity is a colloid particle.
 45. The kit as in claim 41, wherein thesignaling entity is not a colloid particle.
 46. The colloid particle asin claim 41, further comprising a colloid particle, wherein thesignaling entity is attached to the colloid particle.
 47. A compositioncomprising: at least a portion of a shed cell surface receptorinterchain binding region; and a signaling entity immobilized relativeto or adapted to be immobilized relative to the portion.
 48. A kitcomprising: a species able to bind to a portion of a cell surfacereceptor that remains attached to the cell surface after shedding of acell surface receptor interchain binding region; and a signaling entityimmobilized relative to or adapted to be immobilized relative to thespecies.
 49. A kit as in claim 48, wherein the cell surface receptor isMUC1.
 50. The kit as in claim 48, wherein the signaling entity is acolloid particle.
 51. The kit as in claim 48, wherein the signalingentity is not a colloid particle.
 52. The kit particle as in claim 48,further comprising a colloid particle, wherein the signaling entity isattached to the colloid particle.
 53. The kit as in claim 48, whereinthe species able to bind to a portion of a cell surface receptor thatremains attached to the cell surface after shedding of a cell surfacereceptor interchain binding region is a protein of about 17 kD.
 54. Thekit as in claim 49, wherein the species able to bind to a portion of acell surface receptor that remains attached to the cell surface aftershedding of a cell surface receptor interchain binding region is aprotein of about 23 kD.
 55. The kit as in claim 49, wherein the speciesable to bind to a portion of a cell surface receptor that remainsattached to the cell surface after shedding of a cell surface receptorinterchain binding region is a protein of about 35 kD
 56. The kit as inclaim 49, wherein the species able to bind to a portion of a cellsurface receptor that remains attached to the cell surface aftershedding of a cell surface receptor interchain binding region containssequences derived from the protein 14-3-3
 57. The kit as in claim 49,wherein the portion comprises 14-3-3.
 58. The kit as in claim 49,wherein the portion comprises cathepsin D.
 59. The kit as in claim 49,wherein the portion comprises NM23.
 60. The kit as in claim 49, whereinthe portion comprises Human annexin V.
 61. The kit as in claim 49,wherein the species able to bind to a portion of a cell surface receptorthat remains attached to the cell surface after shedding of a cellsurface receptor interchain binding region contains at least onesequence derived from beta-lipotropin.
 62. The kit as in claim 49,wherein the species able to bind to a portion of a cell surface receptorthat remains attached to the cell surface after shedding of a cellsurface receptor interchain binding region is a cleavage product ofproopiomelanocortin.
 63. A kit comprising: a species able to bind to aportion of a cell surface receptor that includes the interchain bindingregion; and a signaling entity immobilized relative to or adapted to beimmobilized relative to the species.
 64. A kit as in claim 63, whereinthe cell surface receptor is MUC1.
 65. The kit as in claim 63, whereinthe signaling entity is a colloid particle.
 66. The kit as in claim 63,wherein the signaling entity is not a colloid particle.
 67. The colloidparticle as in claim 63, further comprising a colloid particle, whereinthe signaling entity is attached to the colloid particle.
 68. A peptidespecies comprising: at least a fragment of a sequence that correspondsto that portion of a cell surface receptor that interacts with anactivating ligand such as a growth factor to promote cell proliferation,the portion being detached from any cell; and an affinity tag.
 69. Apeptide species as in claim 68, wherein the affinity tag is connected tothe fragment.
 70. A peptide species as in claim 68, wherein the affinitytag defines a portion of a continuous amino acid sequence that includesboth the fragment and the affinity tag.
 71. The species of claim 68,wherein the affinity tag is a polyamino acid tag.
 72. The species ofclaim 68, wherein the affinity tag is a polyhistidine tag.
 73. Thespecies of claim 68, wherein the affinity tag is a GST tag.
 74. Thespecies of claim 68, wherein the affinity tag is biotin.
 75. The speciesof claim 68, wherein the affinity tag is Thioredoxin.
 76. The species ofclaim 68, wherein the affinity tag is selected to bind to a speciesimmobilizied with respect to the surface of an article.
 77. The speciesof claim 68, further comprising an article having a surface, and aspecies able to capture the affinity tag immobilizied with respect tothe surface.
 78. The species of claim 68, wherein the article is aparticle.
 79. The species of claim 68, wherein the affinity tag isfastened to the C-terminus of the portion of the receptor.
 80. Thespecies of claim 68, wherein the cell surface receptor is MUC1.
 81. Thespecies of claim 68, wherein the cell surface receptor portion comprises12 or more contiguous amino acids in the sequenceGTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPSAQSGA (SEQ ID NO: 7)
 82. Apeptide species as in claim 68, wherein the fragment comprises at leasta portion of PSMGFR.
 83. A peptide species as in claim 68, wherein thefragment comprises PSMGFR.
 84. A peptide species as in claim 68, whereinthe fragment comprises at least a fragment of the sequence thatcorresponds to that portion of MUC1 that interacts with an activatingligand such as a growth factor to promote cell proliferation inassociation with MUC1-dependent tumorigenesis.
 85. A peptide species asin claim 68, wherein the fragment comprises enough of the sequence thatcorresponds to that portion of MUC1 that remains attached to the cellsurface after shedding of the cell surface receptor interchain bindingregion in association with MUC1-dependent tumorigenesis such that abiomolecule that interacts with that portion of MUC1 that remainsattached to the cell surface after shedding of the cell surface receptorinterchain binding region in association with MUC1-dependenttumorigenesis interacts with the fragment.
 86. A kit comprising: aparticle; and at least a fragment of the sequence that corresponds tothat portion of a cell surface receptor that interacts with anactivating ligand such as a growth factor to promote cell proliferation,the fragment being detached from any cell, fastened to or adapted to befastened to the particle.
 87. The kit of claim 86, wherein the cellsurface receptor is MUC1.
 88. A kit comprising: an article having asurface; and a biomolecule that binds to a portion of a cell surfacereceptor that interacts with an activating ligand such as a growthfactor to promote cell proliferation, the biomolecule being fastened toor adapted to be fastened to the surface of the article.
 89. The kit ofclaim 88, wherein the article comprises a particle.
 90. The kit of claim88, wherein the cell surface receptor is MUC1.
 91. The kit of claim 88,further comprising: a second particle; and a portion of a cell surfacereceptor that remains attached to the cell surface after shedding of thecell surface receptor interchain binding region, the portion beingdetached from any cell, fastened to or adapted to be fastened to thesecond particle.
 92. The kit as in claim 88, wherein the biomoleculethat binds to a portion of a cell surface receptor that remains attachedto the cell surface after shedding of a cell surface receptor interchainbinding region is a protein of about 17 kD.
 93. The kit as in claim 88,wherein the biomolecule that binds to a portion of a cell surfacereceptor that remains attached to the cell surface after shedding of acell surface receptor interchain binding region is a protein of about 23kD.
 94. The kit as in claim 88, wherein the biomolecule that binds to aportion of a cell surface receptor that remains attached to the cellsurface after shedding of a cell surface receptor interchain bindingregion is a protein of about 35 kD.
 95. The kit as in claim 88, whereinthe biomolecule that binds to a portion of a cell surface receptor thatremains attached to the cell surface after shedding of a cell surfacereceptor interchain binding region contains sequences derived from theprotein 14-3-3.
 96. The kit as in claim 88, wherein the biomolecule thatbinds to a portion of a cell surface receptor that remains attached tothe cell surface after shedding of a cell surface receptor interchainbinding region contains sequences derived from Cathepsin D.
 97. The kitas in claim 88, wherein the biomolecule that binds to a portion of acell surface receptor that remains attached to the cell surface aftershedding of a cell surface receptor interchain binding region containssequences derived from NM23.
 98. The kit as in claim 88, wherein thebiomolecule that binds to a portion of a cell surface receptor thatremains attached to the cell surface after shedding of a cell surfacereceptor interchain binding region contains sequences derived from humanannexin V.
 99. The kit as in claim 88, wherein the biomolecule thatbinds to a portion of a cell surface receptor that remains attached tothe cell surface after shedding of a cell surface receptor interchainbinding region contains at least one sequence derived frombeta-lipotropin.
 100. The kit as in claim 88, wherein the biomoleculethat binds to a portion of a cell surface receptor that remains attachedto the cell surface after shedding of a cell surface receptor interchainbinding region is a cleavage product of proopiomelanocortin.
 101. Thekit as in claim 88, wherein the biomolecule that binds to a portion of acell surface receptor that remains attached to the cell surface aftershedding of a cell surface receptor interchain binding region isselected from the group which includes calcimycin, fusaric acid,L-α-methyl-dopa, and etomoxir.
 102. The kit as in claim 88, wherein thebiomolecule that binds to a portion of a cell surface receptor thatremains attached to the cell surface after shedding of a cell surfacereceptor interchain binding region comprises calcimycin.
 103. The kit asin claim 88, wherein the biomolecule that binds to a portion of a cellsurface receptor that remains attached to the cell surface aftershedding of a cell surface receptor interchain binding region comprisesfusaric acid.
 104. The kit as in claim 88, wherein the biomolecule thatbinds to a portion of a cell surface receptor that remains attached tothe cell surface after shedding of a cell surface receptor interchainbinding region comprises L-α-methyl-dopa.
 105. The kit as in claim 88,wherein the biomolecule that binds to a portion of a cell surfacereceptor that remains attached to the cell surface after shedding of acell surface receptor interchain binding region comprises etomoxir. 106.The composition of claim 88, wherein the biomolecule is derived from acell line selected from the group consisting of HTB-133, CRL-1504, a ndCRL-1500.
 107. A method comprising: exposing a ligand capable of bindingwith a portion of a cell surface receptor that remains attached to thecell surface after shedding of a cell surface receptor interchainbinding region, and an agent capable of blocking said binding, to acandidate drug for disruption of interaction between the ligand and theagent; and determining disruption of the interaction by the candidatedrug.
 108. A method comprising: exposing a portion of a cell surfacereceptor that remains attached to the cell surface after shedding of acell surface receptor interchain binding region which is capable ofbinding with a ligand, and an agent capable of blocking said binding, toa candidate drug for disruption of interaction between the portion andthe agent; and determining disruption of the interaction by thecandidate drug.
 109. A method comprising: exposing a synthetic drug, anda biological target of the synthetic drug, to a candidate drug which mayinteract with the biological target to a degree greater than theinteraction between the synthetic drug and the target; and determiningdisruption of the interaction by the candidate drug.
 110. A method as inclaim 109, wherein the synthetic drug is a derivative of fusaric acid.111. A method as in claim 109, wherein the synthetic drug is aderivative of L-α-methyl-dopa.
 112. A method as in claim 109, whereinthe synthetic drug is a derivative of etomoxir.
 113. A method fortreating a subject having a cancer characterized by the aberrantexpression of MUC1, comprising: administering to the subject fusaricacid in an amount effective to reduce tumor growth.
 114. A method as inclaim 113, wherein the subject is otherwise free of symptoms calling fortreatment with calcimycin.
 115. A method as in claim 113, wherein themethod comprises administering to the subject fusaric acid in an amounteffective to block the interaction of a natural ligand and the portionof the MUC1 receptor that remains attached to the cell surface aftershedding of the cell surface receptor interchain binding region.
 116. Amethod as in claim 113, wherein the method comprises administering tothe subject fusaric acid in an amount effective to reduce shedding ofthe interchain binding region of the MUC1 receptor.
 117. The method of113, wherein the levels of shed interchain binding region are reducedrelative to a level measured in a past sample.
 118. The method of 113,wherein the levels of shed interchain binding region are reducedrelative to a control sample.
 119. A method for treating a subjecthaving a cancer characterized by the aberrant expression of MUC1,comprising: administering to the subject etomoxir in an amount effectiveto reduce tumor growth.
 120. A method as in claim 119, wherein thesubject is otherwise free of symptoms calling for treatment withetomoxir.
 121. A method as in claim 119, wherein the method comprisesadministering to the subject etomoxir in an amount effective to blockthe interaction of a natural ligand and the portion of the MUC1 receptorthat remains attached to the cell surface after shedding of the cellsurface receptor interchain binding region.
 122. A method as in claim119, wherein the method comprises administering to the subject etomoxirin an amount effective to reduce shedding of the interchain bindingregion of the MUC1 receptor.
 123. The method of 119, wherein the levelsof shed interchain binding region are reduced relative to a levelmeasured in a past sample.
 124. The method of 119, wherein the levels ofshed interchain binding region are reduced relative to a control sample.125. A method for treating a subject having a cancer characterized bythe aberrant expression of MUC1, comprising: administering to thesubject L-α-methyl-dopa in an amount effective to reduce tumor growth.126. A method as in claim 125, wherein the subject is otherwise free ofsymptoms calling for treatment with L-α-methyl-dopa.
 127. A method as inclaim 125, wherein the method comprises administering to the subjectL-α-methyl-dopa in an amount effective to block the interaction of anatural ligand and the portion of the MUC1 receptor that remainsattached to the cell surface after shedding of the cell surface receptorinterchain binding region.
 128. A method as in claim 125, wherein themethod comprises administering to the subject L-α-methyl-dopa in anamount effective to reduce shedding of the interchain binding region ofthe MUC1 receptor.
 129. The method of 125, wherein the levels of shedinterchain binding region are reduced relative to a level measured in apast sample.
 130. The method of 125, wherein the levels of shedinterchain binding region are reduced relative to a control sample. 131.A method for treating a subject having a cancer characterized by theaberrant expression of MUC1, comprising: administering to the subjectcalcimycin in an amount effective to reduce tumor growth.
 132. A methodas in claim 131, wherein the subject is otherwise free of symptomscalling for treatment with calcimycin.
 133. A method as in claim 131,wherein the method comprises administering to the subject calcimycin inan amount effective to block the interaction of a natural ligand and theportion of the MUC1 receptor that remains attached to the cell surfaceafter shedding of the cell surface receptor interchain binding region.134. A method as in claim 131, wherein the method comprisesadministering to the subject calcimycin in an amount effective to reduceshedding of the interchain binding region of the MUC1 receptor.
 135. Themethod of 134, wherein the levels of shed interchain binding region arereduced relative to a level measured in a past sample.
 136. The methodof 134, wherein the levels of shed interchain binding region are reducedrelative to a control sample.
 137. A method for treating a subjecthaving a cancer characterized by the aberrant expression of MUC1,comprising: administering to the subject butylindazole in an amounteffective to reduce tumor growth.
 138. A method as in claim 137, whereinthe subject is otherwise free of symptoms calling for treatment withbutylindazole.
 139. A method as in claim 137, wherein the methodcomprises administering to the subject butylindazole in an amounteffective to block the interaction of a natural ligand and the portionof the MUC1 receptor that remains attached to the cell surface aftershedding of the cell surface receptor interchain binding region.
 140. Amethod as in claim 137, wherein the method comprises administering tothe subject butylindazole in an amount effective to reduce shedding ofthe interchain binding region of the MUC1 receptor.
 141. The method of137, wherein the levels of shed interchain binding region are reducedrelative to a level measured in a past sample.
 142. The method of 137,wherein the levels of shed interchain binding region are reducedrelative to a control sample.
 143. A method for treating a subjecthaving a cancer characterized by the aberrant expression of MUC1,comprising: administering to the subject NS1619 in an amount effectiveto reduce tumor growth.
 144. A method as in claim 143, wherein thesubject is otherwise free of symptoms calling for treatment with NS1619.145. A method as in claim 143, wherein the method comprisesadministering to the subject NS1619 in an amount effective to block theinteraction of a natural ligand and the portion of the MUC1 receptorthat remains attached to the cell surface after shedding of the cellsurface receptor interchain binding region.
 146. A method as in claim143, wherein the method comprises administering to the subject NS1619 inan amount effective to reduce shedding of the interchain binding regionof the MUC1 receptor.
 147. The method of 143, wherein the levels of shedinterchain binding region are reduced relative to a level measured in apast sample.
 148. The method of 143, wherein the levels of shedinterchain binding region are reduced relative to a control sample. 149.A method comprising: exposing a composition selected among calcimycin,butylindazone, NS1619, fusaric acid, L-α-methyl-dopa, and etomoxir, anda biomolecule that binds to a portion of a cell surface receptor thatremains attached to the cell surface after shedding of a cell surfacereceptor interchain binding region, to a candidate drug which mayinterfere with interaction between the composition and the biomolecule;and determining disruption of the interaction by the candidate drug.150. A method of treating a subject having cancer or at risk fordeveloping cancer comprising: administering to the subject an agent thatreduces cleavage of a cell surface receptor.
 151. A method of treating asubject having cancer or at risk for developing cancer comprising:administering to the subject an agent that reduces cleavage of a cellsurface receptor interchain binding region from the cell surface. 152.The method of claim 150, wherein the cell surface receptor is MUC1. 153.The method of claim 151, wherein the interchain binding region comprisesa contiguous amino acid sequence of at least 12 amino acids from thesequence GFLGLSNIKFRPGSVVVQLTLAFRE.
 154. The method of claim 150,wherein the interchain binding region comprises a contiguous amino acidsequence of about 12 to 18 amino acids, within the region of the humanMUC1 receptor amino acids 507 through 549 (refers to Spicer et alsequence—corresponds to amino acids 1067 through 1100 of Genbankaccession # PI5941, PID G547937).
 155. The method of claim 150, whereinthe interchain binding region comprises a contiguous amino acid sequenceof about 12 to 18 amino acids, within the region of the human MUC1receptor amino acids 525 through 549 (refers to Spicer et alsequence—corresponds to amino acids 1085 through 1109 of Genbankaccession # PI5941, PID G547937).
 156. The method of claim 150, whereinthe cancer is selected from the group consisting of: breast, prostate,lung ovarian, colorectal, and brain cancer.
 157. The method of claim150, wherein the cancer is characterized by the aberrant expression ofthe MUC1 receptor.
 158. A method comprising: determining an amount ofcleavage of a cell surface receptor interchain binding region from acell surface; and evaluating indication of cancer or potential forcancer based upon the determining step.
 159. A method as in claim 158,wherein the cell surface receptor is MUC1.
 160. A method as in claim158, comprising diagnosing cancer in a subject by determining an amountof shed cell surface receptor interchain binding region in a subjectsample; and evaluating indication of cancer or potential for cancerbased upon the determining step.
 161. A method as in claim 158, whereinthe evaluating step comprises correlating the amount in a sample to anamount in a control as an indication of cancer or potential for cancer.162. A method as in claim 158, comprising: determining an amount of cellsurface receptor interchain binding region at the surface of a cell froma subject; and evaluating indication of cancer or potential for cancerbased upon the determining step.
 163. The method of claim 158, whereinthe interchain binding region comprises a contiguous amino acid sequenceof at least 12 amino acids from the sequence GFLGLSNIKFRPGSVVVQLTLAFRE.164. The method of claim 158, wherein the interchain binding regioncomprises a contiguous amino acid sequence of about 12 to 18 aminoacids, within the region of the human MUC1 receptor amino acids 507through 549 (refers to Spicer et al sequence—corresponds to amino acids1067 through 1100 of Genbank accession # PI5941, PID G547937).
 165. Themethod of claim 158, wherein the interchain binding region comprises acontiguous amino acid sequence of about 12 to 18 amino acids, within theregion of the human MUC1 receptor amino acids 525 through 549 (refers toSpicer et al sequence-corresponds to amino acids 1085 through 1109 ofGenbank accession # PI5941, PID G547937).
 166. The method of claim 160,wherein the sample is a fluid sample.
 167. The method of claim 160,wherein the sample is blood.
 168. The method of claim 160, wherein thesample is a tissue sample.
 169. The method of claim 160, wherein thesample is a proliferating cell line derived from a subject's cells. 170.The method of claim 158, wherein the cancer is characterized by aberrantexpression of MUC1.
 171. The method of claim 158, wherein the amount ofinterchain binding region is determined by a method selected from thegroup consisting of MALDI, western blotting, PCR, LCR, rtPCR, cyclingprobe technology, gel electrophoresis, or antibody-based assay, magneticcell sorting, flourescence activated cell sorting, bead-based assays oran ELISA assay.
 172. The method of claim 158, wherein the amount ofinterchain binding region is determined by an aggregation assay. 173.The method of claim 158, wherein the amount of interchain binding regionis determined by a colloid-based method such as colloid-colloid orcolloid-bead assay.
 174. The method of claim 158, wherein the sample isselected from the group consisting of: a needle biopsy, a tissuespecimen, a tissue surface in an intraoperative procedure, and a tissuesurface or cellular solution in a minimally invasive procedure such as alaparoscopy.
 175. A method comprising: determining a site of cleavage ofa cell surface receptor in a sample from a subject; and evaluating anindication of cancer or potential for cancer based upon the determiningstep.
 176. The method of claim 175, wherein the cell surface receptor isMUC1.
 177. The method of claim 175, wherein the sample is selected fromthe group consisting of: a needle biopsy, a tissue specimen, a tissuesurface in an intraoperative procedure, and a tissue surface or cellularsolution in a minimally invasive procedure such as a laparoscopy. 178.The method of claim 175, wherein the sample is a fluid sample.
 179. Themethod of claim 175, wherein the sample is blood.
 180. The method ofclaim 175, wherein the sample is a tissue sample.
 181. The method ofclaim 175, wherein the cancer is selected from the group consisting of:breast, prostate, lung, ovarian, colorectal, and brain cancer.
 182. Amethod as in claim 175, wherein the cancer is characterized by theaberrant expression of MUC1.
 183. The method of claim 175, wherein thesite of cleavage is determined by a method selected from the groupconsisting of MALDI, western blotting, PCR, LCR, rtPCR, cycling probetechnology, gel electrophoresis, or antibody-based assay, magnetic cellsorting, flourescence activated cell sorting, bead-based assays or anELISA assay.
 184. The method of claim 175, wherein the amount ofinterchain binding region is determined by a colloid-based method suchas colloid-colloid or colloid-bead assay.
 185. A method of determining acleavage site of a cell surface comprising: contacting a cell with anagent that binds specifically to one potential cell surface receptorcleavage site and another agent that binds specifically to anotherpotential cell surface receptor cleavage site; and comparing the ratioof binding of the two agents to the cell surface.
 186. The method ofclaim 185, wherein the surface cell receptor is MUC1.
 187. A method ofdiagnosing a physiological state indicative of cancer or potential forcancer, comprising determining a specific cleavage state of MUC1distinguishable from a different cleavage state of MUC1.
 188. A methodcomprising: determining a first amount of cleavage of a cell surfacereceptor interchain binding region from a cell surface of a sample froma subject; determining a second amount of cleavage of a cell surfacereceptor interchain binding region from a cell surface of a sample fromthe subject; comparing the first amount to the second amount.
 189. Amethod as in claim 188, comprising comparing the first amount to thesecond amount as an indication of progression of and/or effectiveness oftreatment for cancer.
 190. A method as in claim 188, comprisingcomparing the first amount to the second amount as an indication foradministration of an agent for prevention of cancer.
 191. A method as inclaim 188, wherein the subject is undergoing treatment for cancer, themethod comprising comparing the first amount to the second amount as anindication of effectiveness of the treatment.
 192. A method as in claim188, wherein the cell surface receptor is MUC1.
 193. The method of claim192, wherein the interchain binding region comprises a contiguous aminoacid sequence of at least 12 amino acids from the sequenceGFLGLSNIKFRPGSVVVQLTLAFRE.
 194. The method of claim 192, wherein theinterchain binding region comprises a contiguous amino acid sequence ofabout 12 to 18 amino acids, within the region of the human MUC1 receptoramino acids 507 through 549 (refers to Spicer et al sequence—correspondsto amino acids 1067 through 1100 of Genbank accession # PI5941, PIDG547937).
 195. The method of claim 188, wherein the interchain bindingregion comprises a contiguous amino acid sequence of about 12 to 18amino acids, within the region of the human MUC1 receptor amino acids525 through 549 (refers to Spicer et al sequence-corresponds to aminoacids 1085 through 1109 of Genbank accession # PI5941, PID G547937).196. The method of claim 188, wherein the sample is a fluid sample. 197.The method of claim 188, wherein the sample is blood.
 198. The method ofclaim 188, wherein the sample is a tissue sample.
 199. The method ofclaim 188, wherein the cancer is selected from the group consisting of:breast, prostate, lung, ovarian, colorectal, and brain cancer.
 200. Themethod of claim 188, wherein the sample is a proliferating cell linederived from a patient's cells.
 201. The method of claim 188, whereinthe amount of interchain binding region is determined by a methodselected from the group consisting of MALDI, western blotting, PCR, LCR,rtPCR, cycling probe technology, gel electrophoresis, or antibody-basedassay, magnetic cell sorting, flourescence activated cell sorting,bead-based assays or an ELISA assay.
 202. The method of claim 188,wherein the amount of interchain binding region is determined by anaggregation assay.
 203. The method of claim 188, wherein the amount ofinterchain binding region is determined by a colloid-based method suchas colloid-colloid or colloid-bead assay.
 204. The method of claim 188,wherein the sample is selected from the group consisting of: a needlebiopsy, a tissue specimen, a tissue surface in an intraoperativeprocedure, and a tissue surface or cellular solution in a minimallyinvasive procedure such as a laparoscopy.
 205. The method of claim 188,wherein the amount of interchain binding region is determined by acolloid-based method such as colloid-colloid or colloid-bead assay. 206.The method of claim 188, wherein the sample is selected from the groupconsisting of: a needle biopsy, a tissue specimen, a tissue surface inan intraoperative procedure, and a tissue surface or cellular solutionin a minimally invasive procedure such as a laparoscopy.
 207. A methodas in claim 188, comprising determining a first amount of a cell surfacereceptor interchain binding region at the surface of a cell in a samplefrom a subject, determining a second amount of a cell surface receptorinterchain binding region at the surface of a cell in a sample from thesubject, comparing the first amount to the second amount.
 208. A methodas in claim 188, comprising determining a first amount of a shed cellsurface receptor interchain binding region in a sample from a subject,determining a second amount of a shed cell surface receptor interchainbinding region in a sample from the subject, comparing the first amountto the second amount.